PASylated VEGFR/PDGFR fusion proteins and their use in therapy

ABSTRACT

A protein comprising (i) a domain of the Platelet-Derived Growth Factor receptor (PDGFR) and (ii) a domain of the Vascular Endothelial Growth Factor receptor (VEGFR) is provided. In a preferred embodiment, said domain of PDGFR and said domain of VEGFR are attached by a linker consisting of proline, alanine and serine. The domain of PDGFR and said domain of VEGFR can also be attached by a linker consisting of proline and alanine. Compositions comprising the proteins, as well as therapeutic uses thereof are also provided.

RELATED APPLICATIONS

The present application claims priority under 37 U.S.C. § 371 to International Patent Application No. PCT/CN2018/115733, filed Nov. 15, 2018, which claims priority to and the benefit of European Patent Application No. 17204968.6, filed on Dec. 1, 2017, and Chinese Patent Application No. 201711136582.6, filed on Nov. 16, 2017. The contents of these applications are hereby incorporated by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, is named 028622-0317_SL_v3.txt and is 306 kb in size.

FIELD OF THE INVENTION

The present invention relates to a protein comprising (i) a domain of the Platelet-Derived Growth Factor receptor (PDGFR) and (ii) a domain of the Vascular Endothelial Growth Factor receptor (VEGFR). In a preferred embodiment, said domain of PDGFR and said domain of VEGFR are attached by a linker consisting of proline, alanine and serine. The domain of PDGFR and said domain of VEGFR can also be attached by a linker consisting of proline and alanine. The present invention also provides compositions comprising the proteins, as well as therapeutic uses thereof.

BACKGROUND OF THE INVENTION

Current state-of-the-art anti-angiogenic therapies target the VEGF pathway, which is the main essential signaling pathway for angiogenesis, including pathological angiogenesis in cancer and eye diseases. However, the long-term outcomes of anti-VEGF monotherapy in patients with eye diseases are somewhat disappointing (Dugel, 2013) since chronic anti-VEGF treatment seems to induce disease-resistance in some patient populations, which is often associated with a substantial loss of vision (Rofagha et al., 2013; Ying et al., 2014; Rosenfeld et al., 2011). As the VEGF level varies in the vitreous of patients, it was proposed that those poor responders of anti-VEGF therapy may need much higher doses of anti-VEGF drugs due to higher levels of VEGF. However, 1-year results from the Clinical Study READ-3 showed no additional benefit of using 4 times higher dose than the regular regimen (Nguyen et al., 2014; Bai et al, 2016). Such observations suggest that multiple pathways are involved in retinal and choroidal neovascularization in eye diseases. In fact, growing clinical and laboratory evidence indicates that, in addition to VEGF, which is a key player in neovascular (or wet) age-related macular degeneration (AMD; Rosenfeld et al., 2006; Heier et al., 2012), PDGF may also play a crucial role in the pathogenesis of this disease. In fact, dual inhibition of VEGF and PDGF may be more effective than targeting VEGF alone (Robins et al., 1994; Benjamin et al., 1998; Zehetner et al., 2014; Bergers et al., 2003; Erber et al., 2004; Pachydaki et al., 2012).

The pathological condition AMD occurs when unstable and highly permeable blood vessels grow and invade from the choroid into the retina, where leakage and bleeding results in a rapid loss of sight (during a period of a few weeks to months). In this setting, VEGF is one of the most potent inducers of vascular permeability known (Carmeliet, 2000), even though the precise mechanisms by which VEGF increases vascular permeability are not entirely clear.

Studies examining postnatal remodeling of the retina provided initial clues as to the importance of VEGF and PDGF in wet AMD (Benjamin et al., 1998) while work on cancer models gave the final push to pursue anti-VEGF/PDGF combination therapies for the treatment of wet AMD (Bergers et al., 2003; Erber et al., 2004). Patients under anti-VEGF monotherapy typically gain an initial improvement in visual acuity (i.e., clarity of vision) in the first 3 or 4 months of treatment, which is followed by a plateau that persists throughout the study (Dugel, 2013). In these first months of treatment, Anti-VEGF monotherapy acts primarily on fenestrated endothelial cells that form the inner lining of the vessel wall, causing a decrease in edema and, consequently, initial improvement in visual acuity. Thereafter, however, the remainder of the neovascular complex seems to be protected by pericyte cells that coat and stabilize the endothelial tube. In this situation, pericytes are thought to render the survival of blood vessels resistant to VEGF inhibition, which may account for the plateau that is usually observed after initial anti-VEGF treatment (Dugel, 2013). Notably, anti-VEGF therapy may not only lead to endothelial cell apoptosis but also enhance pericyte recruitment, thereby potentially reversing the effect of VEGF inhibition (Winkler et al., 2004; Pachydaki et al., 2012).

The arsenal of clinically useful VEGF blockers has evolved over time, with newer generations offering potentially improved anti-angiogenic activity by increasing the affinity towards VEGF-A and/or a number of VEGF isoforms as well as family members they inhibit. In principle, these blockers fall into two categories: (i) monoclonal antibodies, alternative binding proteins (scaffolds) or engineered soluble receptor fragments and (ii) small molecule inhibitors of the kinase domains of VEGFR and related receptors.

One of the first anti-VEGF therapies approved by the FDA for AMD was pegaptanib (Macugen), an RNA aptamer that binds and neutralizes VEGF-A165 (Gragoudas et al., 2004). The first protein-based therapy employing a VEGF-neutralizing strategy was bevacizumab (Avastin, Genentech), a recombinant humanized anti-VEGF antibody designed to block all VEGF isoforms via antigen recognition by its variable region. Bevacizumab was initially approved for the treatment of metastatic colorectal cancer, non-small-cell lung cancer and glioblastoma multiforme (Grothey et al., 2009; Ferrara et al., 2004).

Concomitantly with the development of such cancer therapies, VEGF was found to also play a pivotal role in neovascular AMD and diabetic retinopathy. Starting from this notion, ranibizumab (Lucentis, Genentech) was developed based on bevacizumab as an affinity-matured antigen-binding fragment (Fab) specifically for intravitreal administration to treat vascular eye diseases, especially the wet or neovascular form of AMD (Ferrara et al., 2006) and, recently, also for diabetic retinopathy (Stewart, 2017). The smaller size of the Fab compared with a full size antibody was thought to enhance its diffusion from the vitreous into the retina as well as choroid (Ferrara et al., 2006).

VEGF-Trap (aflibercept; Regeneron Pharmaceuticals) is an engineered soluble decoy receptor that binds VEGF-A based on the molecular interaction of the growth factor with its cognate cellular receptors VEGFR-1 and VEGFR-2. VEGF-Trap consists of a fully human amino-acid sequence comprising the second Ig domain of human VEGFR-1 and the third Ig domain of human VEGFR-2 fused in-line with the constant region (Fc) of human IgG1 (Holash et al., 2002). Therefore, VEGF-Trap has a broader specificity than an antibody, recognizing not only multiple isoforms of VEGF-A but also the related VEGF-B, P1GF (placental growth factor), and PIGF2 (Papadopoulos et al., 2012), which all are physiological ligands of the two tyrosine kinase (TK) receptors VEGFR-1 and VEGFR-2.

SUMMARY OF THE INVENTION

Despite proven efficacy and availability of the reagents described so far, additional and more efficacious anti-VEGF therapies are needed in order to improve VEGF targeting and/or to overcome resistance to existing anti-VEGF therapies. Currently, chronic suppression with serial intravitreal injections of VEGF antagonists is often required for maintaining disease control, while none of the available medications causes complete regression of the choroidal neovascular membrane. In line with this, not all patients respond to treatment, with some developing into non-responders. Ideally, new approaches would address these limitations of current (mono)therapy.

One of the key factors responsible for resistance to VEGF blockage, either intrinsically or adapted during treatment, is the redundancy in the VEGF signalling system (Giuliano & Pages, 2013). An increase in the expression of other proangiogenic factors may possibly fuel alternate signaling pathways for angiogenesis, which could trigger VEGF-independent neovascularization and cause resistance to mono anti-VEGF drugs. The amalgamation of drugs that specifically address more than one pathologenic pathway could potentially enhance the efficacy of therapy by targeting key pathways in a characteristically synergistic or an additive manner.

Apart from the VEGF axis, PDGFs and PDGFRs are validated therapeutic targets in a variety of diseases, especially cancer and vascular disorders (Andrae et al., 2008). PDGFs are hetero- or homodimers of A and B polypeptide chains or homodimers of C or D chains that interact with their cognate PDGF receptors: all PDGF versions except for PDGF-DD bind the PDGFR-α receptor whereas only PDGF-BB and PDGF-DD bind the PDGF-β receptor (Hoch et al., 2003). Thus, compared to PDGFR-β, PDGFR-α possesses broader ligand-binding activity and, furthermore, higher affinity for both PDGF-AA and PDGF-BB as well as, in particular, PDGF-CC.

To date, the basis for such distinct specificities remains unclear. PDGF-CC has been shown to be relevant in both choroidal and retinal neovascularization (Hou et al., 2010; Cao et al., 2002). A pathogenic role for PDGF-BB was implicated in ischemic retinopathies such as proliferative diabetic retinopathy, proliferative vitreoretinopathy, and choroidal neovascularization. Notably, during the processes of angiogenesis and vessel maturation the recruitment of pericytes to the growing endothelial tube is regulated by platelet-derived growth factor-B (PDGF-BB) via signalling through the PDGF receptor β (PDGFR-β). In a pre-clinical rabbit model of proliferative retinopathy, intraocular injection of PDGF-BB-inhibiting aptamers was shown to protect the eye against retinal detachment (Akiyama et al. 2006).

Notably, preliminary results from clinical trials using intravitreal injection of PDGF-blocking agents in conjunction with intravitreal anti-VEGF therapy have demonstrated the potential of combining both strategies also for the treatment of AMD (Diago et al., 2008; Boyer et al., 2009).

However, addressing both types of growth factors at once in a clinical setting still faces technical difficulties. Recently, clinical phase II and phase III studies of Fovista (E10030; Ophthotech), an anti-PDGF-BB pegylated aptamer, were evaluated as an adjunct to ranibizumab. Although the first results from these trials partially demonstrated a benefit of co-administration of E10030 with ranibizumab to patients suffering from wet AMD (Jaffe et al., 2016), ocular adverse events were more frequently reported in the combination therapy group receiving both drugs by separate intravitreal injections.

In another phase II clinical trail on wet AMD, the co-formulation of the two antibody-like molecules aflibercept (VEGF trap) and rinucumab (anti-PDGFR-β antibody, known as REGN2176-3), being administered via single injection was related to more adverse events compared to the aflibercept monotherapy. Patients receiving the combination suffered from increase in conjunctival haemorrhage, eye irritation and eye pain: 23.5% and 20% for the two combination groups versus 16% for aflibercept alone. (CAPELLA; ClinicalTrials.gov Identifier: NCT02418754).

Of note, both drugs, aflibercept and rinucumab, contain crystallizable fragments (Fc) of IgG while it is not known if the Fc component, also known as immunological effector domain, affects physiological mechanisms in the eye. Normally, a physical barrier, the retina-blood-barrier, prevents the free entry of immunoglobulins (Igs) and other large macromolecules into and out of the eye, thus establishing an immune-priviliged microenvironment which makes this organ immunologically unique. Although the fate of high concentrations of therapeutic Ig-based drugs after intravitreal injection is not well understood, evidence exists that the Fc component interacts with retinal Fc receptors and therefore may contribute to an intraretinal inflammatory response in AMD (Souid et al., 2016; Powner et al., 2014; Murinello et al., 2014).

Ideally, protein-based drugs intended for chronical ocular disease treatment should offer extended intraocular half-life to allow less frequent dosing, as each injection procedure constitutes a significant burden for patients and entails a risk of complications (Day et al., 2011). For such a protein drug one way of gaining half-life extension is genetic fusion with a polypeptide that provides a desirable pharmacokinetic profile but is otherwise (physiologically and biochemically) inert. This approach further allows the design of stable second-generation protein drugs with two or more fusion partners, each comprising a unique targeting modality.

Thus, the technical problem underlying the present invention is the provision of means and methods for a therapy targeting both VEGF and ligands of PDGFR.

The technical problem is solved by provision of the embodiments characterized in the claims.

Accordingly, the present invention relates to a protein comprising

-   (i) a domain of the Platelet-Derived Growth Factor receptor (PDGFR);     and -   (ii) a domain of the Vascular Endothelial Growth Factor receptor     (VEGFR).

In a preferred aspect, a protein is provided herein, the protein comprising

-   (i) an extracellular domain of the human Platelet-Derived Growth     Factor receptor (PDGFR); and -   (ii) an extracellular domain of the human Vascular Endothelial     Growth Factor receptor (VEGFR).

In a preferred embodiment, said domain of PDGFR and said domain of VEGFR are attached by a linker consisting of proline, alanine and serine.

As explained herein below, the synergistic effect of VEGF and PDGF signaling inhibition can be mediated by one therapeutic protein. As illustrated in the examples, single-chain proteins were designed that are capable of binding to VEGF and PDGF ligands at the same time. This type of fusion protein functions as a molecular trap for VEGFs, PDGFs and related ligands and is therefore beneficial in pathological processes where these ligands act synergistically, including AMD or cancer.

As shown in the examples, these types of proteins were designed as fusion between the N-terminal ectodomains of VEGF receptors 1 and 2 as well as PDGF receptor α (PDGFR-α), which are involved in ligand-binding of VEGF or PDGF, respectively. Both classes of receptors (VEGFR-1/2 and PDGFR-α) encompass a very broad ligand-binding activity, which is thought to be beneficial in a disease state where angiogenesis is the dominating process. Like all protein-tyrosine kinase receptors, VEGFR-1, VEGFR-2 and PDGFR-α (and also PDGFR-β) consist of an extracellular region of five to seven Ig-like domains (D1-D7), a single transmembrane segment and an intracellular split catalytic tyrosine kinase domain (Shibuya et al., 1999; Stuttfeld et al., 2009). Binding of the dimeric VEGF/PDGF ligands to these receptors generally occurs at the second and third Ig-like domains (D2, D3), where it promotes homo- or heterodimerization of the receptor and, consequently, signal transduction. The proximal domains 4-7 (D4-7) of the extracellular region seem to be important for stabilizing the ligand-receptor complex, whereas the domain closest to the cell membrane, D7, is crucial for ligand-induced tyrosine phosphorylation and cell signaling.

Thus, for the construction of an effective decoy receptor fragment it is, as shown herein, adequate to utilize predominantly domains from the N-terminal extracellular region which are directly involved in binding of the ligands.

In the examples, the extracellular moiety of VEGFR was placed at the C-terminal end of the fusion protein and has the same composition as the high affinity ligand-binding region of the engineered hybrid VEGFR1-D2/VEGFR2-D3 ectodomains described in U.S. Pat. No. 5,952,199. The PDGFR-α moiety, comprising the first three ectodomains D1-3 of the receptor, was arranged at the N-terminal end of the fusion protein, thus preserving the natural N-terminus of PDGFR-α, including its signal peptide which gets processed upon secretion.

Although not much is known about the molecular structure of PDGFR-α, structures of the related PDGFR-13 and VEGFR receptors (Schlessinger, 2000; Shim et al., 2010) provide information about the central part of the corresponding ligand/receptor recognition complexes, which is perceived to be generally similar since PDGFs and VEGFs are of common evolutionary origin (McDonald and Hendrickson, 1993). From these structures it is known that the D1 domains of PDGFR-β and VEGFRs are not directly involved in ligand binding but, because of a hydrophobic interface between D1 and D2, serve as a cap for the ligand-binding D2 domains (Hye-Ryong et al., 2010; Leppanen et al., 2013). Therefore, inclusion of the first domain D1 in the decoy version of PDGFR-α was considered herein beneficial for the therapeutic fusion protein as provided herein in the examples. In fact, the presence of D1 in PDGFR-α seems to have also a small differential effect on ligand binding to PDGF-AA, as learnt from deletion analysis within the ectodomain of PDGF-α (Mahadewan et al., 1995).

In a preferred aspect, a fusion protein is provided, wherein the extracellular parts D1-3 of PDGFR-α and D2/D3 of VEGFR1/2 are linked by a PAS-polypeptide sequence or, alternatively, a Ser-free P/A sequence. Such PAS/PA sequences are for example disclosed in WO2008/155134 A1 and WO2011/144756 A1. The PAS/PA spacer provides structural flexibility to the individual ectodomains, thus allowing access of both VEGF and PDGF ligands. In addition, these random coil sequences greatly increase the hydrodynamic volume of the fusion protein, which slows down clearance of the fused ectodomains in vivo and, thus, prolongs and/or enhances the pharmacological effect (Schlapschy et al., 2013). In addition, PAS/PA polypeptides are hydrophilic homo-polymers of the small natural L-amino acids proline, alanine and serine (or proline and alanine, respectively), which provides biocompatibility and facilitates metabolization.

The random coil nature of the PAS linker/spacer sequence (Schlapschy et al., 2013) provides the individual VEGFR and PDGFR ectodomains with high flexibility such that, in the presence of ligands, each arm of the decoy receptor fusion is able to bind to the dimeric ligand (growth factor), eventually forming a functional decoy dimer (see FIG. 2 ). Once formed via complex formation with the first ligand, either VEGF or PDGF, such a dimerized fusion protein further gains functional affinity for the second ligand via the avidity effect. Hence, the affinities of PDGFR and VEGFR ectodomains should synergistically sum up by way of multiple binding interactions, especially in a disease condition where both ligands are abundant.

This is highly advantageous, as in the dimerized ectodomain receptor fusion, if the first ligand is present, ideally VEGF, the relatively moderate affinity of natural PDGFR ectodomains for their homo/heterodimeric PDGF ligands can be boosted by the high affinity ligand-binding site of the hybrid VEGFR1-D2/VEGFR2-D3 domains towards VEGF-A (Holash et al., 2002). The decoy receptor as provided and disclosed herein should be comparable with the corresponding membrane-bound natural receptors in terms of affinity and specificity on the one hand, but it should be incapable of triggering signaling, or presenting the agonist to signaling receptor complexes, on the other hand.

In accordance with the above, the examples demonstrate that exemplary proteins provided herein

-   -   Inhibit VEGF₁₆₅-induced HUVEC cell proliferation (Example 20);     -   Inhibit the intersegmental vessels (ISVs) development in         zebrafish embryos (Example 21);     -   Inhibit the tumor neovascularization induced by human VEGFA         (Example 22);     -   Show an increased half-life (T½) in SD rats (Example 23);     -   Inhibit laser-induced Choroidal Neovascularization (CNV) in         cynomolgus monkeys (Examples 24);     -   Show an increased half-life (T½) in New Zealand rabbits (Example         25);     -   Show complex formation with target compounds in native PAGE and         electromobility gel shift assay (Example 26);     -   Inhibit VEGF₁₆₅-induced HUVEC cell proliferation (Example 27).

In view of the herein demonstrated properties of the proteins, they can be advantageously used in a therapeutic setting as disclosed herein.

The present invention relates to the following items:

-   1. Protein comprising     -   (i) an extracellular domain of the human Platelet-Derived Growth         Factor receptor (PDGFR); and     -   (ii) an extracellular domain of the human Vascular Endothelial         Growth Factor receptor (VEGFR). -   2. The protein of item 1, wherein said domain of PDGFR and said     domain of VEGFR are attached by a linker consisting of proline,     alanine and serine. -   3. The protein of item 2, wherein said proline residues constitute     more than 4% and less than 40% of said linker. -   4. The protein of item 2 or 3, wherein said linker has an amino acid     sequence as follows: (ASPAAPAPASPAAPAPSAPA (SEQ ID NO: 71)) n,     wherein n is an integer of 10-100. -   5. The protein of item 4, wherein said linker comprising an amino     acid sequence as follows: (ASPAAPAPASPAAPAPSAPA (SEQ ID NO: 71)) n,     wherein n is an integer of 10-60. -   6. The protein of item 5, wherein said linker comprising an amino     acid sequence as follows: CASPAAPAPASPAAPAPSAPA (SEQ ID NO: 71)) n,     wherein n is an integer of 10-40. -   7. The protein of item 6, wherein said linker comprising an amino     acid sequence as follows: (ASPAAPAPASPAAPAPSAPA (SEQ ID NO: 71)) n,     wherein n is an integer of 10-30. -   8. The protein of item 7, wherein said linker comprising an amino     acid sequence as follows: (ASPAAPAPASPAAPAPSAPA (SEQ ID NO: 71)) n,     wherein n is 10, 20 or 30. -   9. The protein of item 4, wherein said linker has an amino acid     sequence as shown in SEQ ID No. 2 or wherein said linker is a     polypeptide encoded by a nucleic acid molecule having a nucleotide     sequence as shown in SEQ ID No. 1. -   10. The protein of item 1, wherein said domain of PDGFR and said     domain of VEGFR are attached by a linker consisting of proline and     alanine. -   11. The protein of item 10, wherein said proline residues constitute     more than about 10% and less than about 75% of said linker. -   12. The protein of item 10 or 11, wherein said linker has an amino     acid sequence as follows: (AAPAAPAPAAPAAPAAPA (SEQ ID NO: 72)) n,     wherein n is an integer of 10-100. -   13. The protein of item 12, wherein said linker has an amino acid     sequence as shown in SEQ ID No. 70 or wherein said linker is a     polypeptide encoded by a nucleic acid molecule having a nucleotide     sequence as shown in SEQ ID No. 69. -   14. The protein of any one of items 2 to 10, wherein said linker has     an amino acid sequence consisting of about 50 to about 3000 amino     acid residues. -   15. The protein of any one of item 14, wherein said domain of PDGFR     comprises one or more of Ig domains 1 to 5 of PDGFR. -   16. The protein of any one of item 15, wherein said domain of PDGFR     comprises one or more of Ig domains 1 to 3 of PDGFR. -   17. The protein of any one of items 1 to 16, wherein said domain of     PDGFR comprises Ig domains 1 to 3 of PDGFR. -   18. The protein of any one of items 1 to 17, wherein said domain of     PDGFR is capable of binding to Platelet-Derived Growth Factor     (PDGF). -   19. The protein of item 18, wherein said PDGF is a PDGF dimer. -   20. The protein of items 19, wherein said PDGF dimer is a PDGF     homodimer or a PDGF heterodimer. -   21. The protein of any one of items 1 to 20, wherein said PDGFR is     human PDGFRα. -   22. The protein of any one of items 1 to 21, wherein said domain of     PDGFR comprises:     -   (a) a protein having an amino acid sequence as shown in SEQ ID         No. 4 or SEQ ID No. 20;     -   (b) a protein as defined in (a) wherein 1 to 10 amino acids are         deleted, inserted, added or substituted;     -   (c) a polypeptide encoded by a nucleic acid molecule having a         nucleotide sequence as shown in SEQ ID No. 3 or SEQ ID No. 19;     -   (d) a polypeptide having an amino acid sequence encoded by a         nucleic acid hybridizing under stringent conditions to the         complementary strand of nucleic acid molecules as defined in         (c);     -   (e) a polypeptide having at least 70% identity to the         polypeptide of any one of (a) to (d); and     -   (f) a polypeptide having an amino acid sequence encoded by a         nucleic acid being degenerate as a result of the genetic code to         the nucleotide sequence of a nucleic acid as defined in (c) or         (d). -   23. The protein of item 21 or 22, wherein said domain of PDGFR is     capable of binding to Platelet-Derived Growth Factor (PDGF), wherein     said PDGF is a PDGF homodimer, and wherein said PDGF homodimer is a     PDGFA homodimer, a PDGFB homodimer, or a PDGFC homodimer. -   24. The protein of item 21 or 22, wherein said domain of PDGFR is     capable of binding to Platelet-Derived Growth Factor (PDGF), wherein     said PDGF preferably is a PDGF heterodimer, and wherein said PDGF     heterodimer preferably is a heterodimer of PDGFAB. -   25. The protein of any one of items 1 to 20, wherein said PDGFR is     human PDGFRβ3. -   26. The protein of any one of items 1 to 20 and 25, wherein said     domain of PDGFR comprises:     -   (a) a protein having an amino acid sequence as shown in SEQ ID         No.6;     -   (b) a protein as defined in (a) wherein 1 to 10 amino acids are         deleted, inserted, added or substituted;     -   (c) a polypeptide encoded by a nucleic acid molecule having a         nucleotide sequence as shown in SEQ ID No. 5;     -   (d) a polypeptide having an amino acid sequence encoded by a         nucleic acid hybridizing under stringent conditions to the         complementary strand of nucleic acid molecules as defined in         (c);     -   (e) a polypeptide having at least 70% identity to the         polypeptide of any one of (a) to (d); and     -   (f) a polypeptide having an amino acid sequence encoded by a         nucleic acid being degenerate as a result of the genetic code to         the nucleotide sequence of a nucleic acid as defined in (c) or         (d). -   27. The protein of item 25 or 26, wherein said domain of PDGFR is     capable of binding to Platelet-Derived Growth Factor (PDGF), wherein     said PDGF preferably is a PDGF homodimer, and wherein said PDGF     homodimer preferably is a PDGFB homodimer. -   28. The protein of any one of items 18 to 27, wherein said     Platelet-Derived Growth Factor (PDGF) is human PDGF. -   29. The protein of any one of items 1 to 28, wherein said domain of     VEGFR comprises one or more of Ig domains 1 to 7 of VEGFR. -   30. The protein of any one of items 1 to 29, wherein said domain of     VEGFR comprises Ig domain 2 and/or Ig domain 3 of VEGFR. -   31. The protein of any one of items 1 to 30, wherein said domain of     VEGFR comprises Ig domain 2 and Ig domain 3 of VEGFR. -   32. The protein of any one of items 1 to 31, wherein said VEGFR is     human VEGFR-1 or human VEGFR-2. -   33. The protein of any one of items 1 to 32, wherein said domain of     VEGFR comprises Ig domain 2 of VEGFR-1 and Ig domain 3 of VEGFR-2. -   34. The protein of any one of items 1 to 33, wherein said domain of     VEGFR comprises     -   (a) a protein having an amino acid sequence as shown in SEQ ID         No. 8;     -   (b) a protein as defined in (a) wherein 1 to 10 amino acids are         deleted, inserted, added or substituted;     -   (c) a polypeptide encoded by a nucleic acid molecule having a         nucleotide sequence as shown in SEQ ID No. 7;     -   (d) a polypeptide having an amino acid sequence encoded by a         nucleic acid hybridizing under stringent conditions to the         complementary strand of nucleic acid molecules as defined in         (c);     -   (e) a polypeptide having at least 70% identity to the         polypeptide of any one of (a) to (d); and     -   (f) a polypeptide having an amino acid sequence encoded by a         nucleic acid being degenerate as a result of the genetic code to         the nucleotide sequence of a nucleic acid as defined in (c) or         (d). -   35. The protein of any one of items 1 to 34, wherein said domain of     VEGFR is capable of binding to Vascular Endothelial Growth Factor     (VEGF). -   36. The protein of item 35, wherein said Vascular Endothelial Growth     Factor (VEGF) is a VEGF dimer. -   37. The protein of item 36, wherein said VEGF dimer is a VEGF     homodimer. -   38. The protein of item 37, wherein said VEGF homodimer is a VEGFA     homodimer. -   39. The protein of any one of items 35 to 38, wherein said Vascular     Endothelial Growth Factor (VEGF) is human VEGF. -   40. The protein of any one of items 1 to 39, wherein said protein is     a fusion protein. -   41. The protein of any one of items 1 to 40, wherein said protein     comprises:     -   (a) a protein having an amino acid sequence as shown in SEQ ID         No. 16, SEQ ID No. 46, SEQ ID No. 48, SEQ ID No. 50, SEQ ID No.         52, SEQ ID No. 54, SEQ ID No. 56, SEQ ID No. 58, SEQ ID No. 60,         SEQ ID No. 62, SEQ ID No. 64, SEQ ID No. 66 or SEQ ID No. 68;     -   (b) a protein as defined in (a) wherein 1 to 10 amino acids are         deleted, inserted, added or substituted;     -   (c) a polypeptide encoded by a nucleic acid molecule having a         nucleotide sequence as shown in SEQ ID No. 15, SEQ ID No. 45,         SEQ ID No. 47, SEQ ID No. 49, SEQ ID No. 51, SEQ ID No. 53, SEQ         ID No. 55, SEQ ID No. 57, SEQ ID No. 59, SEQ ID No. 61, SEQ ID         No. 63, SEQ ID No. 65 or SEQ ID No. 67;     -   (d) a polypeptide having an amino acid sequence encoded by a         nucleic acid hybridizing under stringent conditions to the         complementary strand of nucleic acid molecules as defined in         (c);     -   (e) a polypeptide having at least 70% identity to the         polypeptide of any one of (a) to (d); and     -   (f) a polypeptide having an amino acid sequence encoded by a         nucleic acid being degenerate as a result of the genetic code to         the nucleotide sequence of a nucleic acid as defined in (c) or         (d). -   42. The protein of any one of items 1 to 41, wherein said protein     comprises an N-terminal signal polypeptide sequence. -   43. The protein of item 42, wherein said N-terminal signal     polypeptide sequence is the N-terminal signal polypeptide sequence     of PDGFR. -   44. The protein of item 43, wherein said N-terminal signal     polypeptide sequence is the N-terminal signal polypeptide sequence     of human PDGFRα. -   45. The protein of any one of items 42 to 44, wherein said     N-terminal signal polypeptide sequence has an amino acid sequence as     shown in SEQ ID No. 10 or wherein said N-terminal signal polypeptide     sequence is a polypeptide encoded by a nucleic acid molecule having     a nucleotide sequence as shown in SEQ ID No. 9. -   46. The protein of any one of items 1 to 45, wherein said protein     further comprises a purification tag. -   47. The protein of item 46, wherein said purification tag is a     His-tag. -   48. The protein of item 46 or 47, wherein said purification tag has     an amino acid sequence as shown in SEQ ID No. 12 or wherein said     purification tag is a polypeptide encoded by a nucleic acid molecule     having a nucleotide sequence as shown in SEQ ID No. 11. -   49. The protein of any one of items 1 to 48, wherein the protein is     arranged from N-terminus to C-terminus in the order:     -   (optional signal sequence)-one or more domains of         PDGFR-(optional linker)-one or more domains of VEGFR-(optional         purification tag) or     -   (optional signal sequence)-one or more domains of         VEGFR-(optional linker)-one or more domains of PDGFR-(optional         purification tag) or     -   (optional signal sequence)-(optional linker)-one or more domains         of VEGFR-one or more domains of PDGFR-(optional purification         tag) or     -   (optional signal sequence)-(optional linker)-one or more domains         of PDGFR-one or more domains of VEGFR-(optional purification         tag) or     -   (optional signal sequence)-(optional linker)-one or more domains         of PDGFR-(optional linker)-one or more domains of         VEGFR-(optional linker)-(optional purification tag). -   50. The protein of any one of items 1 to 49, wherein the protein is     arranged from N-terminus to C-terminus in the order:     -   (optional signal sequence)-one or more domains of         PDGFR-(PAS/PA)-one or more domains of VEGFR-(optional         purification tag) or     -   (optional signal sequence)-one or more domains of         VEGFR-(PAS/PA)-one or more domains of PDGFR-(optional         purification tag) or     -   (optional signal sequence)-(PAS/PA)-one or more domains of         VEGFR-one or more domains of PDGFR-(optional purification tag)         or     -   (optional signal sequence)-(PAS/PA)-one or more domains of         PDGFR-one or more domains of VEGFR-(optional purification tag)         or     -   (optional signal sequence)-(PAS/PA)-one or more domains of         PDGFR-(PAS/PA)-one or more domains of VEGFR-(PAS/PA)-(optional         purification tag). -   51. The protein of any one of items 1 to 50, wherein the protein is     arranged from N-terminus to C-terminus in the order:     -   (optional signal sequence)-one or more domains of PDGFR-(GGGGS         (SEQ ID NO: 73))n-(PAS/PA) -(GGGGS (SEQ ID NO: 73))n-one or more         domains of VEGFR-(optional purification tag) or     -   (optional signal sequence)-one or more domains of VEGFR-(GGGGS         (SEQ ID NO: 73))n-(PAS/PA) -(GGGGS (SEQ ID NO: 73))n-one or more         domains of PDGFR-(optional purification tag);     -   wherein, n=0-5. -   52. The protein of any one of items 1 to 51, wherein said protein     comprises     -   (a) a protein having an amino acid sequence as shown in SEQ ID         No. 14, SEQ ID No. 22, SEQ ID No. 24, SEQ ID No. 26, SEQ ID No.         28, SEQ ID No. 30, SEQ ID No. 32, SEQ ID No. 34, SEQ ID No. 36,         SEQ ID No. 38, SEQ ID No. 40, SEQ ID No. 42 or SEQ ID No. 44;     -   (b) a protein as defined in (a) wherein 1 to 10 amino acids are         deleted, inserted, added or substituted;     -   (c) a polypeptide encoded by a nucleic acid molecule having a         nucleotide sequence as shown in SEQ ID No. 13, SEQ ID No. 15,         SEQ ID No. 21, SEQ ID No. 23, SEQ ID No. 25, SEQ ID No. 27, SEQ         ID No. 29, SEQ ID No. 31, SEQ ID No. 33, SEQ ID No. 35, SEQ ID         No. 37, SEQ ID No. 39, SEQ ID No. 41 or SEQ ID No. 43,     -   (d) a polypeptide having an amino acid sequence encoded by a         nucleic acid hybridizing under stringent conditions to the         complementary strand of nucleic acid molecules as defined in         (c);     -   (e) a polypeptide having at least 70% identity to the         polypeptide of any one of (a) to (d); and     -   (f) a polypeptide having an amino acid sequence encoded by a         nucleic acid being degenerate as a result of the genetic code to         the nucleotide sequence of a nucleic acid as defined in (c) or         (d). -   53. A nucleic acid molecule encoding the protein of any one of items     1 to 52. -   54. A vector comprising the nucleic acid of item 53. -   55. A host cell comprising the nucleic acid of items 53 or the     vector of item 54. -   56. The host cell according to item 55, wherein said host cell is a     eukaryotic host cell or a prokaryotic host cell. -   57. The host cell according to item 56, wherein said prokaryotic     host cell is E. coli. -   58. The host cell according to item 56, wherein said eukaryotic host     cell is a fungal or animal cell. -   59. The host cell according to item 58, wherein said animal cell is     a HEK cell or a CHO cell. -   60. A method for the preparation of a protein of any one of items 1     to 52. -   61. The method of item 60, comprising culturing the host cell     according to any one of items 55 to 59 and isolating said protein     from the culture or from said cell. -   62. A composition comprising the protein of any one of items 1 to     52, the protein prepared by the method of item 60 or 61, the nucleic     acid of item 53, the vector of item 54, or the cell of any one of     items 55 to 58. -   63. The composition according to item 62 which is a pharmaceutical     composition, optionally further comprising (a) pharmaceutical     acceptable carrier(s). -   64. The protein of any one of items 1 to 52, the protein prepared by     the method of item 60 or 61, the nucleic acid of item 53, the vector     of item 54, the cell of any one of items 55 to 58, or the     composition of item 62 or 63, for use as a medicament. -   65. The protein of any one of items 1 to 52, the protein prepared by     the method of item 60 or 61, the nucleic acid of item 53, the vector     of item 54, the cell of any one of items 55 to 58, or the     composition of item 62 or 63, for use in the treatment of ophthalmic     diseases, cancer, renal fibrosis, cirrhosis, arthosclerosis, portal     hypertension or systemic sclerosis. -   66. The protein for use of item 65, the nucleic acid for use of item     65, the vector for use of item 65, the cell for use of item 65, or     the composition for use of item 65, wherein said cancer is a solid     cancer. -   67. The protein for use of item 66, the nucleic acid for use of item     66, the vector for use of item 66, the cell for use of item 66, or     the composition for use of item 66, wherein said solid cancer is     colon cancer, hepatocellular carcinoma, non-small cell lung cancer,     soft tissue sarcoma, prostate cancer, breast cancer, ovarian cancer,     glioma, dermatofibrosarcoma protuberans, oral squamous cell     carcinoma, pancreatic cancer. -   68. The protein for use of item 65, the nucleic acid for use of item     65, the vector for use of item 65, the cell for use of item 65, or     the composition for use of item 65, wherein said cancer is a     non-solid cancer. -   69. The protein for use of item 68, the nucleic acid for use of item     68, the vector for use of item 68, the cell for use of item 68, or     the composition for use of item 68, wherein said non-solid cancer is     leukemia or non-Hodgkin's lymphoma. -   70. The protein for use of item 65, the nucleic acid for use of     claim 65, the vector for use of claim 65, the cell for use of claim     65, or the composition for use of claim 65, wherein said ophthalmic     diseases is age-related macular degeneration (AMD), Diabetic     retinopathy (DR), Diabetic macular edema (DME), Choroidal     neovascularization (CNV), Retinal vein occlusion (RVO), Central     retinal vein occlusion (CRVO), Branch retinal vein occlusion (BRVO),     or pathologic myopia (PM). -   71. The protein for use of item 65, the nucleic acid for use of     claim 65, the vector for use of claim 65, the cell for use of claim     65, or the composition for use of claim 65, wherein said ophthalmic     diseases is age-related macular degeneration (AMID).

In certain aspects, the following items are provided herein:

As mentioned above, a protein is provided herein, the protein comprising

-   -   (i) an extracellular domain of the human Platelet-Derived Growth         Factor receptor (PDGFR); and     -   (ii) an extracellular domain of the human Vascular Endothelial         Growth Factor receptor (VEGFR).

As indicated above, said domain of PDGFR and said domain of VEGFR are, in a preferred embodiment, attached by a linker consisting of proline, alanine and serine.

The Platelet-Derived Growth Factor (PDGF) family consists of disulphide-bonded homodimers of A-, B-, C- and D-polypeptide chains, and the heterodimer PDGF-AB. PDGF isoforms are reported to exert their cellular effects by binding to their respective receptors (PDGF receptors (PDGFR)). The terms “Platelet-Derived Growth Factor”, “PDGF”, “Platelet-Derived Growth Factor protein” and “PDGF protein” are used interchangeably herein. The terms “Platelet-Derived Growth Factor receptor”, “PDGF receptor”, “PDGFR”, “Platelet-Derived Growth Factor receptor protein”, “PDGF receptor protein” and “PDGFR protein” are used interchangeably herein.

Vascular Endothelial Growth Factor (VEGF) and their receptors (VEGFR) are reported to regulate both vasculogenesis (the development of blood vessels from precursor cells during early embroygenesis) and angiogenesis (the formation of blood vessles from pre-existing vessels at a later stage). The VEGF family of genes contains at least 7 members, whereas the VEGFR family of genes has 3 to 4 members depending on the vertebrate species. The terms “Vascular Endothelial Growth Factor”, “VEGF”, “Vascular Endothelial Growth Factor protein” and “VEGF protein” are used interchangeably herein. The terms “Vascular Endothelial Growth Factor receptor”, “VEGF receptor”, “VEGFR”, “Vascular Endothelial Growth Factor receptor protein”, “VEGF receptor protein” and “VEGFR protein” are used interchangeably herein.

The meaning of the term “domain” or “protein domain” is well known in the art and the terms are used accordingly herein. The terms “domain” and “protein domain” are used interchangeably herein. A protein domain can be viewed as the basic structural unit of a protein structure. The core of each domain is usually largely composed of a set of interconnected β sheets or α helices or both. Domains are usually constructed from a section of a polypeptide chain that contains normally between 50 to 350 amino acids.

It is envisaged that the proteins provided herein can act as a “decoy” receptor, i.e. that they can bind to the ligand PDGF and/or VEGF.

In a preferred aspect, the domain of PDGFR is capable of binding to Platelet-Derived Growth Factor (PDGF). The PDGF can be a monomer, but is preferably a PDGF dimer. The PDGF dimer can be a PDGF homodimer or a PDGF heterodimer.

In a preferred aspect, the domain of VEGFR is capable of binding to Vascular Endothelial Growth Factor (VEGF). The VEGF can be a monomer, but is preferably a PDGF dimer. The VEGF dimer can be a VEGF homodimer, like a VEGFA homodimer.

More preferably, both the domain of PDGFR is capable of binding to Platelet-Derived Growth Factor (PDGF) and the domain of VEGFR is capable of binding to Vascular Endothelial Growth Factor (VEGF).

The terms “capable of binding”, “binding capacity” and the like are used herein in accordance with the common meaning in the art. In context of ligand-receptor-interactions, “binding capacity” refers to the capacity of a ligand (here PDGF and VEGF respectively) to bind to its receptor (here the domain of PDGFR and domain of VEGFR, respectively).

Ligand binding can be characterized by the IC₅₀ (the concentration of a ligand at which half of the receptor binding sites are occupied).

Binding affinity can be determined using a radio labeled (tagged) ligand, known as a tagged ligand. Non-labelled methods include surface plasmon resonance, dual polarization interferometry, Multi-Parametric Surface Plasmon Resonance (MP-SPR) and Microscal thermophoresis.

PDGF binds normally to the extracellular domain of its receptor PDGFR.

It is preferred herein that the domain of PDGFR comprises or consists of the extracellular domain of PDGFR. The extracellular domain of PDGFR contains 5 Ig-like domains. The term “Ig-like domain” and “Ig domain” are used interchangeably herein. Ligand binding is thought to occur preferentially through Ig domains 2 and 3.

In accordance with the above, the domain of PDGFR can comprise or consist of one or more of Ig domains 1 to 5 of PDGFR, i.e. one or more of Ig domain 1 of PDGFR, Ig domain 2 of PDGFR, Ig domain 3 of PDGFR, Ig domain 4 of PDGFR, Ig domain 5 of PDGFR. Any combinations thereof, as well as the use of fragments or derivatives of one or more of Ig domains 1 to 5 of PDGFR (and any combinations of one or more Ig domains 1 to 5 of PDGFR and of any fragments or derivatives of one or more of Ig domains 1 to 5 of PDGFR is encompassed herein).

The domain of PDGFR to be used herein can for example comprise or consist of one or more of Ig domains 1 to 3 of PDGFR, i.e. one or more of Ig domain 1 of PDGFR, Ig domain 2 of PDGFR and Ig domain 3 of PDGFR. Any combinations thereof, as well as the use of fragments or derivatives of one or more of Ig domains 1 to 3 of PDGFR (and any combinations of one or more g domains 1 to 3 of PDGFR and of any fragments or derivatives of one or more of Ig domains 1 to 3 of PDGFR is encompassed herein).

As shown in the appended example, a protein comprising Ig domains 1 to 3 of PDGFR is indeed capable of binding to PDGF.

In a preferred aspect, the domain of PDGFR comprises or consists of Ig domains 1 to 3 of PDGFR, particularly preferably of Ig domains 1 to 3 of human PDGFRα.

The use of animal PDGFR (i.e. of animal origin), for example an extracellular domain of PDGFR and/or one or more of Ig domains 1 to 5 of PDGFR) is envisaged herein, for example mammalian PDGFR, e.g. rat, mouse, pig, guinea pig, ape PDGFR and the like. It is preferred herein that the PDGFR is human PDGFR (i.e. of human origin), for example an extracellular domain of human PDGFR and/or one or more of Ig domains 1 to 5 of human PDGFR). The amino acid sequence and nucleotide sequence of human PDGFR is well known in the prior art, see e.g. NCBI Reference Sequence: NP_001334758.1, NP_001334756.1, NP_001334757.1, NP_001341945.1, NP_002600.1

It is envisaged herein that the PDGFR domain herein can be composed of portions/fragments of various PDGFR proteins (or PDGFR isoforms), e.g. portions/fragments of PDGFR proteins (and/or PDGFR isoforms) of different origin, e.g. origin of different animals and/or of human origin. For example, the PDGFR domain herein can be composed of a portion/fragment of a PDGFR protein (including various PDGFR isoforms) of human origin and a portion/fragment of a PDGFR protein (including various PDGFR isoforms) of animal origin, e.g. of rat, mouse, pig, guinea pig, or ape PDGFR protein (including various PDGFR isoforms). It is envisaged herein that the PDGFR domain herein can be composed of portions/fragments of various PDGFR isoforms (e.g. various PDGFR isoforms of human and/or animal origin). For example, the PDGFR domain herein can be composed of portions of various human PDGFR isoforms (e.g. various PDGFR isoforms of human origin), e.g. portions of human PDGFRα and/or human PDGFRβ.

For example, the domain of PDGFR can comprise or consist of e.g. one or more of Ig domains 1 to 5 of PDGFR of e.g. origin of different animals and/or of human origin. For example, the domain of PDGFR can comprise or consist of Ig domain 1 and/or 2 of PDGFR of animal origin and Ig domain 3 of PDGFR of human origin (or vice versa). For example, the domain of PDGFR can comprise or consist of e.g. one or more of Ig domains 1 to 5 of various (human) PDGFR isoforms e.g. human PDGFRα and/or human PDGFRβ. For example, the domain of PDGFR can comprise or consist of Ig domain 1 and/or 2 of human PDGFRα and Ig domain 3 of human PDGFRβ (or vice versa). For example, the domain of PDGFR can comprise or consist of Ig domain 1 of human PDGFRα and Ig domains 2 and/or 3 of human PDGFRβ (or vice versa).

For example, compositions are envisaged herein that comprise e.g. proteins comprising a PDGFR domain of different origin, e.g. origin of different animals and/or of human origin. For example, compositions are envisaged that comprise e.g. proteins comprising a PDGFR domain of human origin and proteins comprising a PDGFR domain of animal origin, e.g. of rat, mouse, pig, guinea pig, or ape PDGFR. For example, compositions are envisaged that comprise e.g. proteins comprising a PDGFR domain of various PDGFR isoforms (e.g. various human PDGFR isoforms), like a composition comprising e.g. a protein comprising a PDGFR domain of human PDGFRα and comprising a protein comprising a VEGFR domain of human PDGFRβ.

In a herein preferred aspect, the PDGFR is human PDGFRα.

The domain of PDGFR can comprise or consist of:

-   (a) a protein having an amino acid sequence as shown in SEQ ID No. 4     or SEQ ID No. 20; -   (b) a protein as defined in (a) wherein 1 to 10 amino acids are     deleted, inserted, added or substituted; -   (c) a polypeptide encoded by a nucleic acid molecule having a     nucleotide sequence as shown in SEQ ID No. 3 or SEQ ID No. 19; -   (d) a polypeptide having an amino acid sequence encoded by a nucleic     acid hybridizing under stringent conditions to the complementary     strand of nucleic acid molecules as defined in (c); -   (e) a polypeptide having at least 70% identity to the polypeptide of     any one of (a) to (d); and -   (f) a polypeptide having an amino acid sequence encoded by a nucleic     acid being degenerate as a result of the genetic code to the     nucleotide sequence of a nucleic acid as defined in (c) or (d).

The protein having an amino acid sequence as shown in SEQ ID No. 4 corresponds to Ig domains 1 to 3 of human PDGFRα. A corresponding nucleic acid molecule encoding such a protein is shown in SEQ ID No. 3.

The protein having an amino acid sequence as shown in SEQ ID No. 20 corresponds to Ig domains 1 to 3 of human PDGFRα. A corresponding nucleic acid molecule encoding such a protein is shown in SEQ ID No. 19.

In a preferred embodiment, the domain of PDGFR can comprise or consist of:

-   (a) a protein having an amino acid sequence as shown in SEQ ID No. 4     or SEQ ID No. 20; or -   (c) a polypeptide encoded by a nucleic acid molecule having a     nucleotide sequence as shown in SEQ ID No. 3 or SEQ ID No. 19.

Particularly if the PDGFR is human PDGFRα (or if the PDGFR domain is the PDGFR domain of human PDGFRα), and if the domain of PDGFR is capable of binding to Platelet-Derived Growth Factor (PDGF), said PDGF can be a PDGF homodimer, for example a PDGFA homodimer, a PDGFB homodimer, or a PDGFC homodimer.

Particularly if the PDGFR is human PDGFRα (or if the PDGFR domain is the PDGFR domain of human PDGFRα), and if the domain of PDGFR is capable of binding to Platelet-Derived Growth Factor (PDGF), said PDGF can be a PDGF heterodimer, for example a heterodimer of PDGF-AB.

It is envisaged herein that the PDGFR herein can be human PDGFRβ (or that the PDGFR domain can be the PDGFR domain of human PDGFRβ).

The domain of PDGFR can comprise or consist of:

-   (a) a protein having an amino acid sequence as shown in SEQ ID No.     6; -   (b) a protein as defined in (a) wherein 1 to 10 amino acids are     deleted, inserted, added or substituted; -   (c) a polypeptide encoded by a nucleic acid molecule having a     nucleotide sequence as shown in SEQ ID No. 5; -   (d) a polypeptide having an amino acid sequence encoded by a nucleic     acid hybridizing under stringent conditions to the complementary     strand of nucleic acid molecules as defined in (c); -   (e) a polypeptide having at least 70% identity to the polypeptide of     any one of (a) to (d); and -   (f) a polypeptide having an amino acid sequence encoded by a nucleic     acid being degenerate as a result of the genetic code to the     nucleotide sequence of a nucleic acid as defined in (c) or (d).

The protein having an amino acid sequence as shown in SEQ ID No. 6 corresponds to Ig domains 1 to 3 of human PDGFRβ. A corresponding nucleic acid molecule encoding such a protein is shown in SEQ ID No. 5.

In a preferred aspect, the domain of PDGFR can comprise or consist of:

-   (a) a protein having an amino acid sequence as shown in SEQ ID No.     6;     or -   (c) a polypeptide encoded by a nucleic acid molecule having a     nucleotide sequence as shown in SEQ ID No. 5.

Particularly if the PDGFR is human PDGFRβ (or if the PDGFR domain is the PDGFR domain of human PDGFRβ), and if the domain of PDGFR is capable of binding to Platelet-Derived Growth Factor (PDGF), said PDGF can be a PDGF homodimer, for example, a PDGF-B homodimer.

The use of animal PDGF (i.e. of animal origin) is envisaged herein, for example mammalian PDGF, e.g. rat, mouse, pig, guinea pig, ape PDGF and the like. It is preferred herein that the PDGF is human PDGF (i.e. of human origin). Also the amino acid sequence and nucleotide sequence of PDGF, such as human PDGF, is well known in the prior art, see e.g. NCBI Reference Sequences NP_002598.4, NP_148983.1, NP_002599, NP_148937 or NP_057289.1.

The protein provided herein comprises a domain of VEGFR.

VEGF binds normally to the extracellular domain of its receptor VEGFR.

It is preferred herein that the domain of VEGFR comprises or consists of the extracellular domain of VEGFR. The extracellular domain of VEGFR contains 7 Ig-like domains. The term “Ig-like domain” and “Ig domain” are used interchangeably herein. Ligand binding is thought to occur preferentially to Ig domains 2 and 3.

In accordance with the above, the domain of VEGFR can comprise or consist of one or more of Ig domains 1 to 7 of VEGFR, i.e. one or more of Ig domain 1 of VEGFR, Ig domain 2 of VEGFR, Ig domain 3 of VEGFR, Ig domain 4 of VEGFR, Ig domain 5 of VEGFR, Ig domain 6 of VEGFR and Ig domain 7 of VEGFR. Any combinations thereof, as well as the use of fragments or derivatives of one or more of Ig domains 1 to 7 of VEGFR (and any combinations of one or more Ig domains 1 to 7 of VEGFR and of any fragments or derivatives of one or more of Ig domains 1 to 7 of VEGFR is encompassed herein).

The domain of VEGFR to be used herein can for example comprise or consist of Ig domains 2 and/or 3 of VEGFR, i.e. Ig domain 2 and/or Ig domain 3 of VEGFR. Any combinations thereof, as well as the use of fragments or derivatives of Ig domain 2 and/or Ig domain 3 of VEGFR (and any combinations of Ig domain 2 and/or Ig domain 3 of VEGFR and of any fragments or derivatives of Ig domain 2 and/or Ig domain 3 of VEGFR of VEGFR) is encompassed herein.

As shown in the appended example, a protein comprising Ig domains 2 and 3 of VEGFR is indeed capable of binding to VEGF.

In a preferred aspect, the domain of VEGFR comprises or consists of Ig domains 2 and 3 of VEGFR.

The use of animal VEGFR (i.e. of animal origin), for example an extracellular domain of VEGFR and/or one or more of Ig domains 1 to 7 of VEGFR) is envisaged herein, for example mammalian VEGFR, e.g. rat, mouse, pig, guinea pig, or ape VEGFR and the like. It is preferred herein that the VEGFR is human VEGFR (i.e. of human origin), for example an extracellular domain of human VEGFR and/or one or more of Ig domains 1 to 7 of human VEGFR). The amino acid sequence and nucleotide sequence of human VEGFR is well known in the prior art, see e.g. NCBI Reference Sequences: NP_002010.2, NP_001153392.1, NP_001153502.1, NP_001153503.1 or NP_002244.1. It is preferred herein that the VEGFR is human VEGFR-1 and/or human VEGFR-2.

It is envisaged herein that the VEGFR domain herein can be composed of portions/fragments of various VEGFR proteins (or VEGFR isoforms), e.g. portions/fragments of VEGFR proteins (and/or VEGFR isoforms) of different origin, e.g. origin of different animals and/or of human origin. For example, the VEGFR domain herein can be composed of (a) portion(s)/fragment(s) of a VEGFR protein (including various VEGFR isoforms) of human origin and (a) portion(s)/fragment(s) of a VEGFR protein (including various VEGFR isoforms) of animal origin, e.g. of rat, mouse, pig, guinea pig, or ape VEGFR protein (VEGFR isoforms). It is also envisaged herein that the VEGFR domain herein can be composed of portions/fragments of various VEGFR isoforms (e.g. various VEGFR isoforms of human and/or animal origin). For example, the VEGFR domain herein can be composed of portions/fragments of various human VEGFR isoforms (e.g. various VEGFR isoforms of human origin), e.g. portions/fragments of human VEGFR-1 or human VEGFR-2.

For example, the domain of VEGFR can comprise or consist of e.g. one or more of Ig domains 1 to 7 of VEGFR of e.g. origin of different animals and/or of human origin. For example, the domain of VEGFR can comprise or consist of Ig domain 2 of VEGFR of animal origin and Ig domain 3 of VEGFR of human origin (or vice versa). For example, the domain of VEGFR can comprise or consist of e.g. one or more of Ig domains 1 to 7, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 2 to 3, of various (human) VEGFR isoforms e.g. of human VEGFR-1 and/or human VEGFR-2. For example, the domain of VEGFR can comprise or consist of Ig domain 1 and/or 2 of human VEGFR-1 and Ig domain 3 of human VEGFR-2 (or vice versa). For example, the domain of VEGFR can comprise or consist of Ig domain 1 of human VEGFR-1 and Ig domains 2 and/or 3 of human VEGFR-2 (or vice versa).

In a preferred aspect, the domain of VEGFR comprises or consists of Ig domain 2 of VEGFR-1 and Ig domain 3 of VEGFR-2. In a particularly preferred aspect, the domain of VEGFR comprises or consists of Ig domain 2 of human VEGFR-1 and Ig domain 3 of human VEGFR-2.

For example, compositions are envisaged herein that comprise e.g. proteins comprising a VEGFR domain of different origin, e.g. origin of different animals and/or of human origin. For example, compositions are envisaged that comprise e.g. proteins comprising a VEGFR domain of human origin and proteins comprising a VEGFR domain of animal origin, e.g. of rat, mouse, pig, guinea pig, or ape VEGFR. For example, compositions are envisaged that comprise e.g. proteins comprising a VEGFR domain of various VEGFR isoforms (e.g. various human VEGFR isoforms), like a composition comprising e.g. a protein comprising a VEGFR domain of human VEGFR-1 and comprising a protein comprising a VEGFR domain of human VEGFR-2.

The domain of VEGFR can comprise or consist of:

-   (a) a protein having an amino acid sequence as shown in SEQ ID No.     8; -   (b) a protein as defined in (a) wherein 1 to 10 amino acids are     deleted, inserted, added or substituted; -   (c) a polypeptide encoded by a nucleic acid molecule having a     nucleotide sequence as shown in SEQ ID No. 7; -   (d) a polypeptide having an amino acid sequence encoded by a nucleic     acid hybridizing under stringent conditions to the complementary     strand of nucleic acid molecules as defined in (c); -   (e) a polypeptide having at least 70% identity to the polypeptide of     any one of (a) to (d); and -   (f) a polypeptide having an amino acid sequence encoded by a nucleic     acid being degenerate as a result of the genetic code to the     nucleotide sequence of a nucleic acid as defined in (c) or (d).

The protein having an amino acid sequence as shown in SEQ ID No. 8 corresponds to Ig domain 2 of human VEGFR-1 and Ig domain 3 of human VEGFR-2. A corresponding nucleic acid molecule encoding such a protein is shown in SEQ ID No. 7.

In a preferred embodiment, the domain of VEGFR can comprise or consist of

-   (a) a protein having an amino acid sequence as shown in SEQ ID No.     8; or -   (c) a polypeptide encoded by a nucleic acid molecule having a     nucleotide sequence as shown in SEQ ID No. 7.

As mentioned, preferably the domain of VEGFR is capable of binding to Vascular Endothelial Growth Factor (VEGF).

Particularly if the VEGFR is human VEGFR (or if the VEGFR domain is the VEGFR domain of human VEGFR), and if the domain of VEGFR is capable of binding to VEGF, said VEGF can be a VEGF dimer, particularly a VEGF homodimer, and preferably a VEGFA homodimer.

The use of animal VEGF (i.e. of animal origin) is envisaged herein, for example mammalian VEGF, e.g. rat, mouse, pig, guinea pig, ape VEGF and the like. It is preferred herein that the VEGF is human VEGF (i.e. of human origin). Also the amino acid sequence and nucleotide sequence of VEGF, such as human VEGF, is well known in the prior art, see e.g. NCBI Reference Sequences: NP_001020537.2, NP_001020538.2, NP_001020539.2, NP_001020540.2, NP_001020541.2, NP_001028928.1, NP_001165093.1, NP_001165094.1, NP_001165095.1, NP_001165096.1, NP_001165097.1, NP_001165098.1, NP_001165099.1, NP_001165100.1, NP_001165101.1, NP_001191313.1, NP_001191314.1, NP_001273973.1, NP_001303939.1 or NP_003367.4.

The domain of PDGFR and the domain of VEGFR can be attached by a linker, like a peptide or polypeptide linker. The linker to be used herein primarily serves the purpose to provide the VEGFR and PDGFR domains with high flexibility so that each domain (each arm of the decoy receptor) is able to bind to the (dimeric) ligand (VEGF and PDGF, respectively). Consequently, a protein dimer can form in the presence of ligands, i.e. a functional decoy dimer can form. Thus, the linker/linker sequences does not necessarily contribute to the biological activity, in particular ligand binding (i.e. binding of VEGF and PDGF, respectively), of the herein provided proteins. The linker is preferably a flexible linker. The peptide or polypeptide linker(s) can be composed of flexible residues, like glycine and/or serine.

The linker can have an amino acid sequence consisting of about 50 to about 3000 amino acid residues, e.g. about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500 2600, 2700, 2800, 2900 or 3000 amino acid residues. In a preferred aspect, the linker has an amino acid sequence consisting of 200 amino acid residues.

In a preferred aspect, said domain of PDGFR and said domain of VEGFR are attached by a linker consisting of proline, alanine and serine. In this aspect, the proline residues can constitute more than 4% and less than 40% of said linker.

Preferably, said linker comprising an amino acid sequence as follows: (ASPAAPAPASPAAPAPSAPA (SEQ ID NO: 71)) n, wherein n=10-100; further preferably, n=10-60; more preferably, n=10-40; further preferably, n=10-30; more preferably, n=10, 20 or 30; particularly preferably, the linker can have an amino acid sequence as shown in SEQ ID NO: 2 or said linker can be a polypeptide encoded by a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID No. 1. The linker can have an amino acid sequence consisting of about 50 to about 3000 amino acid residues.

The domain of PDGFR and the domain of VEGFR can be attached by a linker consisting of proline and alanine. In this aspect, the proline residues can constitute more than about 10% and less than about 75% of said linker. Preferably, said linker has an amino acid sequence as follows: (AAPAAPAPAAPAAPAAPA (SEQ ID NO: 72)) n, wherein n is an integer of 10-100; further preferably, said linker has an amino acid sequence as shown in SEQ ID No. 70 or wherein said linker is a polypeptide encoded by a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID No. 69. The linker can have an amino acid sequence consisting of about 50 to about 3000 amino acid residues.

In a preferred aspect, a protein is provided herein, wherein said protein comprises:

-   (a) a protein having an amino acid sequence as shown in SEQ ID No.     16, SEQ ID No. 46, SEQ ID No. 48, SEQ ID No. 50, SEQ ID No. 52, SEQ     ID No. 54, SEQ ID No. 56, SEQ ID No. 58, SEQ ID No. 60, SEQ ID No.     62, SEQ ID No. 64, SEQ ID No. 66 or SEQ ID No. 68; -   (b) a protein as defined in (a) wherein 1 to 10 amino acids are     deleted, inserted, added or substituted; -   (c) a polypeptide encoded by a nucleic acid molecule having a     nucleotide sequence as shown in SEQ ID No. 15, SEQ ID No. 45, SEQ ID     No. 47, SEQ ID No. 49, SEQ ID No. 51, SEQ ID No. 53, SEQ ID No. 55,     SEQ ID No. 57, SEQ ID No. 59, SEQ ID No. 61, SEQ ID No. 63, SEQ ID     No. 65 or SEQ ID No. 67; -   (d) a polypeptide having an amino acid sequence encoded by a nucleic     acid hybridizing under stringent conditions to the complementary     strand of nucleic acid molecules as defined in (c); -   (e) a polypeptide having at least 70% identity to the polypeptide of     any one of (a) to (d); and -   (f) a polypeptide having an amino acid sequence encoded by a nucleic     acid being degenerate as a result of the genetic code to the     nucleotide sequence of a nucleic acid as defined in (c) or (d).

Preferred herein are proteins termed herein EPS1104P, EPS1107P, EPS1108P and EPS1115P. Their sequences are shown among others in the following table:

Trivial SEQ ID No. Name Construct SEQ ID No. 14 EPS1108P PDGFR_(αD123)-PAS(200)-VEGFR1_(D)2/R2_(D3) SEQ ID No. 22 EPS1103P PDGFRα_(D123)-PAS(300)-VEGFR1_(D2)/R2_(D3) SEQ ID No. 24 EPS1104P PDGFRα_(D123)-PAS(400)-VEGFR1_(D2)/R2_(D3) SEQ ID No. 26 EPS1105P VEGFR1_(D2)/R2_(D3)-PAS(200)- PDGFRα_(D123) SEQ ID No. 28 EPS1106P PDGFRα_(D123)-(GGGGS)₃-PAS(200)- (GGGGS)₃-VEGFR1_(D2)/R2_(D3) SEQ ID No. 30 EPS1107P VEGFR1_(D2)/R2_(D3)-(GGGGS)₃-PAS(200)- (GGGGS)₃-PDGFRα_(D123) SEQ ID No. 32 EPS1109P PAS(200)-VEGFR1_(D2)/R2_(D3)-PDGFRα_(D123) SEQ ID No. 34 EPS1110P PAS(200)-PDGFRα_(D123)-VEGFR1_(D2)/R2_(D3) SEQ ID No. 36 EPS1111P PDGFRβ_(D123)-PAS(200)-VEGFR1_(D2)/R2_(D3) SEQ ID No. 38 EPS1113P PDGFRα_(D123)-PAS(600)-VEGFR1_(D2)/R2_(D3) SEQ ID No. 40 EPS1114P PDGFRα_(D123)-(GGGGS)₃-PAS(600)- (GGGGS)₃-VEGFR1_(D2)/R2_(D3) SEQ ID No. 42 EPS1115P VEGFR1_(D2)/R2_(D3)-(GGGGS)₃-PAS(600)- (GGGGS)₃-PDGFRα_(D123) SEQ ID No. 44 EPS1116P mutantPDGFRα_(D123)-PAS(200)- VEGFR1_(D2)/R2_(D3)

The following further defines the linker consisting of proline, alanine and serine/the linker consisting of proline and alanine. It is envisaged herein that the linker forms a random coil.

As used herein, the term “random coil” relates to any conformation of a polymeric molecule, including amino acid polymers, in particular polypeptides made of L-amino acids, in which the individual monomeric elements that form said polymeric structure are essentially randomly oriented towards the adjacent monomeric element or elements while still being chemically linked. In particular, the encoded polypeptide or amino acid polymer adopting/having/forming “random coil conformation” substantially lacks a defined secondary and tertiary structure. The nature of the encoded polypeptide random coils and their methods of experimental identification are known to the person skilled in the art and have been described in the scientific literature (Cantor (1980) Biophysical Chemistry, 2nd ed., W. H. Freeman and Company, New York; Creighton (1993) Proteins—Structures and Molecular Properties, 2nd ed., W. H. Freeman and Company, New York; Smith (1996) Fold. Des. 1:R95-R106) and patent literature, e.g., WO2011/144756 and WO2008/155134.

The linker as comprised in the herein provided protein can adopt/form a random coil conformation, for example, in aqueous solution and/or at physiological conditions. The term “physiological conditions” is known in the art and relates to those conditions in which proteins usually adopt their native, folded conformation. More specifically, the term “physiological conditions” relates to the environmental biophysical parameters as they are typically valid for higher forms of life and, particularly, for mammals, most preferably human beings. The term “physiological conditions” may relate to the biochemical and biophysical parameters as they are normally found in the body, in particular in body fluids, of mammals and in particular in humans. Said “physiological conditions” may relate to the corresponding parameters found in the healthy body as well as the parameters found under disease conditions or in human patients. For example, a sick mammal or human patient may have a higher, yet “physiological” body temperature (i.e., temperature condition) when said mammal or said human suffers from fever. With respect to “physiological conditions” at which proteins adopt their native conformation/state, the most important parameters are temperature (37° C. for the healthy human body), pH (7.35-7.45 for human blood), osmolarity (280-300 mmol/kg H₂O), and, if necessary, general protein content (66-85 g/l serum).

Yet, the person skilled in the art is aware that at physiological conditions these parameters may vary, e.g. the temperature, pH, osmolarity, and protein content may be different in given body or tissue fluids such as blood, liquor cerebrospinalis, peritoneal fluid and lymph (Klinke (2005) Physiologie, 4th edition, Georg Thieme Verlag, Stuttgart). For example, in the liquor cerebrospinalis the osmolarity may be around 290 mmol/kg H₂O and the protein concentration may be between 0.15 g/l and 0.45 g/l while in the lymph the pH may be around 7.4 and the protein content may be between 3 g/l and 5 g/l. When determining whether a polypeptide linker forms/adopts random coil conformation under experimental conditions, the biophysical parameters such as temperature, pH, osmolarity and protein content may be different from the physiological conditions normally found in vivo. Temperatures between 1° C. and 42° C. or preferably 4° C. to 25° C. may be considered useful to test and/or verify the biophysical properties and biological activity of a polypeptide linker (as comprised in the herein provide protein) under physiological conditions in vitro.

Several buffers, which may include solvents and/or excipients for pharmaceutical compositions, are considered to represent “physiological solutions”/“physiological conditions” in vitro, in particular, in experimental settings, for example in the context of CD measurements or other methods that allow the person skilled in the art to determine the structural properties of a protein/amino acid sequence. Examples of such buffers are, e.g., phosphate-buffered saline (PBS, e.g.: 115 mM NaCl, 4 mM KH₂PO₄, 16 mM Na₂HPO₄ pH 7.4), Tris buffers, acetate buffers, citrate buffers or similar buffers. Generally, the pH of a buffer representing “physiological solution conditions” should lie in a range from 6.5 to 8.5, preferably in a range from 7.0 to 8.0, most preferably in a range from 7.2 to 7.7, and the osmolarity should lie in a range from 10 to 1000 mmol/kg H₂O, more preferably in a range from 50 to 500 mmol/kg H₂O and most preferably in a range from 200 to 350 mmol/kg H₂O. Optionally, the protein content of a physiological solution may lie in a range from 0 to 100 g/l, neglecting the investigated protein adopting random coil conformation itself; furthermore, typical stabilizing proteins may be present, for example human or bovine serum albumin.

The polypeptide linkers provided herein not only form random coil conformation under physiological conditions but, more generally, in aqueous solution; e.g., c.f. WO2011/144756. The term “aqueous solution” is well known in the art. An “aqueous solution” may be a solution with a water (H₂O) content of at least about 20%, of at least about 30%, of at least about 40%, of at least about 50%, of at least about 60%, of at least about 70%, of at least about 80% or of at least about 90% H₂O (weight/weight). Accordingly, the encoded polypeptides provided in the present invention may form random coil conformation in aqueous solution, possibly containing other miscible solvents, or in aqueous dispersions with a wider range of temperatures, pH values, osmolarities or protein content.

It is envisaged herein that the random coil conformation of the polypeptide linker is maintained in pharmaceutical compositions like liquid pharmaceuticals/biologicals or lyophilized pharmaceutical compositions. Preferably, “physiological conditions” are to be used in corresponding buffer systems, solvents and/or excipients. Yet, for example, in lyophilized or dried compositions (like, e.g., pharmaceutical compositions), it is envisaged that the random coil conformation of the herein provided random coil polypeptide linker may transiently not be present and/or cannot be detected. However, said random coil polypeptide/linker will adopt/form its random coil again after reconstitution in corresponding buffers/solutions/excipients/solvents or after administration to the body of a patient or of an animal.

In certain aspects of the present invention, the linker consists of proline, alanine and, optionally, serine, wherein no more than 9 consecutive amino acid residues are identical. The linker adopting random coil conformation may comprise a plurality of amino acid repeats, wherein said “amino acid repeats” mainly or exclusively consist of proline, alanine and, optionally, serine amino acid residues, wherein no more than 9 consecutive amino acid residues are identical. The linker adopting random coil conformation may comprise a plurality of amino acid repeats, wherein said “amino acid repeats” mainly or exclusively consist of proline, alanine and serine amino acid residues, wherein no more than 9 consecutive amino acid residues are identical. The linker adopting random coil conformation may comprise a plurality of amino acid repeats, wherein said “amino acid repeats” mainly or exclusively consist of proline and alanine amino acid residues, wherein no more than 9 consecutive amino acid residues are identical.

In certain aspects, the linker comprises a plurality of amino acid repeats, wherein no more than 8 consecutive amino acid residues are identical and wherein said linker forms a random coil, wherein no more than 7 consecutive amino acid residues are identical and wherein said linker forms a random coil, or wherein no more than 6 consecutive amino acid residues are identical and wherein said linker forms a random coil. Particularly preferably, the linker comprises a plurality of amino acid repeats, wherein no more than 5 consecutive amino acid residues are identical and wherein said linker forms a random coil. More particularly preferably, the linker comprises a plurality of amino acid repeats, wherein no more than 4 consecutive amino acid residues are identical and wherein said linker forms a random coil. Most preferably, the linker comprises a plurality of amino acid repeats, wherein no more than 3 consecutive amino acid residues are identical and wherein said linker forms a random coil.

A non-limiting example of an amino acid repeat consisting exclusively of proline, alanine and serine residues is provided herein below: SEQ ID No. 2.

The linker can consist mainly or exclusively of the three amino acid residues proline (Pro, P), alanine (Ala, A) and, optionally, serine (Ser, S). The term “optionally” as used herein means that the linker either consists mainly or exclusively of proline, alanine and serine or consists mainly or exclusively of proline and alanine. The linker consisting mainly or exclusively of the three amino acid residues proline, alanine and serine is referred to herein as “PAS” linker. The linker consisting mainly or exclusively of the two amino acid residues proline and alanine is referred to herein as “PA” linker. A non-limiting example of linker consisting of proline, alanine and serine is given in SEQ ID No. 2. The term “mainly” as used herein means that preferably at least about 90% or at least about 95% of the encoded amino acids are proline, alanine and, optionally, serine, whereby proline, alanine and serine in sum constitute the majority but may not be the only amino acid residues; therefore, the amino acid sequence of the linker is not necessarily 100% proline, alanine and, optionally, serine. Hence, the linker may also comprise other amino acids than proline, alanine and, optionally, serine as minor constituents as long as the linker forms/adopts/has the random coil conformation. Such a random coil conformation can be easily determined by means and methods described herein. Accordingly, the linker that preferably forms random coil can consist mainly of proline, alanine and, optionally, serine.

In case the linker consists of proline and alanine, said proline residues constitute more than about 10% and less than about 75% of said linker. Accordingly, the linker can consist mainly of proline and alanine, wherein the proline residues constitute more than about 10% and less than 75% of the amino acid sequence. The alanine residues comprise the remaining at least 25% to 90% of the amino acid sequence.

Preferably, the amino acid sequence of the linker (the linker) comprises more than about 10%, preferably more than about 12%, more preferably more than about 14%, 18%, 20%, more preferably more than about 22%, 23%, 24%, or 25%, more preferably more than about 27%, 29%, or 30%, more preferably more than about 32%, 33%, or 34% and most preferably more than about 35% proline residues. The amino acid sequence of the linker (the linker) preferably comprises less than about 75%, more preferably less than 70%, more preferably less than 65%, more preferably less than 60%, more preferably less than 55%, more preferably less than 50% proline residues, wherein the lower values are preferred. Even more preferably, the amino acid sequence of the linker (the linker) comprises less than about 48%, 46%, 44%, 42% proline residues. More preferred are amino acid sequences of the linker (the linker) comprising less than about 41%, 40%, 39% 38%, 37% or 36% proline residues, whereby lower values are preferred. More preferred are amino acid sequences of the linker (the linker) comprising less than about 34%, 32%, or 30%. More preferred are amino acid sequences of the linker (the linker) comprising less than about 28%, 26% or 25%. Most preferably, the amino acid sequences of the linker (the linker) comprise less than about 35% proline residues.

Vice versa, the amino acid sequence of the linker (the linker) preferably comprises less than about 90%, more preferably less than 88%, 86%, 84%, 82% or 80% alanine residues, wherein the lower values are preferred. More preferably, the amino acid sequence of the linker (the linker) comprises less than about 79%, 78%, 77%, 76% alanine residues, whereby lower values are preferred. More preferably, the amino acid sequence of the linker (the linker) comprises less than about 74%, 72%, or 70% alanine residues, whereby lower values are preferred. More preferably, the amino acid sequence of the linker (the linker) comprises less than about 69%, 67%, or 65% alanine residues, whereby lower values are preferred. Most preferably, the amino acid sequence of the linker (the linker) comprises less than about 75% alanine residues. Also preferred herein is an amino acid sequence of the linker (the linker) comprising more than about 25%, preferably more than about 30%, more preferably more than about 35%, more preferably more than about 40%, more preferably more than about 45%, more preferably more than about 50%, more preferably more than about 52%, 54%, 56%, 58% or 59% alanine residues, wherein the higher values are preferred. Even more preferably, the amino acid sequence of the linker (the linker) comprises more than about 60%, 61%, 62%, 63% or 64% alanine residues. More preferably, the amino acid sequence of the linker (the linker) comprises more than about 66%, 67%, 69%, or 70% alanine residues. More preferably, the amino acid sequence of the linker (the linker) comprises more than about 72%, 74%, or 75%, alanine residues. Most preferably the amino acid sequence of the linker (the linker) comprises more than about 65% alanine residues.

Accordingly, the linker may comprise an amino acid sequence consisting of about 25% or 30% proline residues and about 75% or 70%, respectively, alanine residues. Alternatively, the linker may comprise an amino acid sequence consisting of about 35% proline residues and about 65% alanine residues. The term “about X %” as used herein above is not limited to the concise number of the percentage, but also comprises values of 10% to 20% additional or 10% to 20% less residues. For example, the term 10% may also relate to 11% or 12% and to 9% or 8%, respectively.

In case the linker consists of proline, alanine and serine, said proline residues can constitute more than about 4% and less than about 40% of said of the amino acid sequence of the linker (the linker). The alanine and the serine residues constitute the remaining amount of said amino acid sequence of the linker (the linker).

Preferably, the amino acid sequence of the linker (the linker) comprises more than about 4%, preferably more than about 6%, more preferably more than about 10%, more preferably more than about 15%, more preferably more than about 20%, more preferably more than about 22%, 23% or 24%, more preferably more than about 26%, 29%, or 30%, more preferably more than about 31%, 32%, 33%, 34% or 35% and most preferably more than about 25% proline residues. The amino acid sequence of the linker (the linker) preferably comprises less than about 40%, more preferably less than 38%, 35%, 30%, 26% proline residues, wherein the lower values are preferred.

The amino acid sequence of the linker (the linker) preferably comprises less than about 95%, more preferably less than 90%, 86%, 84%, 82% or 80% alanine residues, wherein the lower values are preferred. More preferably, the amino acid sequence of the linker (the linker) comprises less than about 79%, 78%, 77%, 76% alanine residues, whereby lower values are preferred. More preferably, the amino acid sequence of the linker (the linker) comprises less than about 75%, 73%, 71%, or 70% alanine residues, whereby lower values are preferred. More preferably, the amino acid sequence of the linker (the linker) comprises less than about 69%, 67%, 66%, or 65% alanine residues, whereby lower values are preferred. More preferably, the amino acid sequence of the linker (the linker) comprises less than about 64%, 63%, 62%, or 60% alanine residues, whereby lower values are preferred. More preferably, the amino acid sequence of the linker (the linker) comprises less than about 59%, 57%, 56%, or 55% alanine residues, whereby lower values are preferred. More preferably, the amino acid sequence of the linker (the linker) comprises less than about 54%, 53%, or 51%, alanine residues, whereby lower values are preferred. Most preferably, the amino acid sequence of the linker (the linker) comprises less than about 50% alanine residues.

Also preferred herein is an amino acid sequence of the linker (the linker) comprising more than about 10%, preferably more than about 15%, 17%, 19%, or 20%, more preferably more than about 22%, 24%, or 25%, more preferably more than about 27%, 29%, or 30%, more preferably more than about 32%, 34% or 35%, more preferably more than about 37%, 39%, or 40%, more preferably more than about 42%, 44% or 45%, more preferably more than about 46%, 47% or 49% alanine residues, wherein the higher values are preferred. Most preferably, the amino acid sequence comprises more than about 50 alanine residues. As mentioned above, the serine residues comprise the remaining amount of said amino acid sequence. Accordingly, the of the linker (the linker) may comprise an amino acid sequence consisting of about 35% proline residues, about 50% alanine and 15% serine residues. The term “about X %” as used herein above is not limited to the concise number of the percentage, but also comprises values of 10% to 20% additional or 10% to 20% less residues. For example, the term 10% may also relate to 11% or 12% or to 9% and 8%, respectively.

However, as mentioned above and further detailed herein below the amino acid sequence of the linker (the linker) may also comprise additional amino acids differing from proline, alanine and, optionally, serine as minor constituents. As already discussed herein above, said minor constituent(s), i.e. amino acid(s) different from proline, alanine or, optionally, serine, may comprise less than about 10% or less than about 5% of the of the linker.

The skilled person is aware that the linker may also form random coil conformation when other residues than proline, alanine and, optionally, serine are comprised as a minor constituent in said amino acid sequence of the linker (the linker). The term “minor constituent” as used herein means that maximally 5% or maximally 10% amino acid residues are different from proline, alanine or serine in the linker. This means that maximally 10 of 100 amino acids may be different from proline, alanine and, optionally, serine, preferably maximally 8%, i.e. maximally 8 of 100 amino acids may be different from proline, alanine and, optionally, serine, more preferably maximally 6%, i.e. maximally 6 of 100 amino acids may be different from proline, alanine and, optionally, serine, even more preferably maximally 5%, i.e. maximally 5 of 100 amino acids may be different from proline, alanine and, optionally, serine, particularly preferably maximally 4%, i.e. maximally 4 of 100 amino acids may be different from proline, alanine and, optionally, serine, more particularly preferably maximally 3%, i.e. maximally 3 of 100 amino acids may be different from proline, alanine and, optionally, serine, even more particularly preferably maximally 2%, i.e. maximally 2 of 100 amino acids may be different from proline, alanine and, optionally, serine and most preferably maximally 1%, i.e. maximally 1 of 100 of the amino acids that are comprised in the random coil polypeptide may be different from proline, alanine and, optionally, serine. Said amino acids different from proline, alanine and, optionally, serine may be selected from the group consisting of Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Tyr, and Val, including posttranslationally modified amino acids or non-natural amino acids (see, e.g., Budisa (2004) Angew Chem Int Ed Engl 43:6426-6463; Young (2010) J Biol Chem 285:11039-11044; Liu (2010) Annu Rev Biochem 79:413-444; Wagner (1983) AngewChem Int Ed Engl 22:816-828; Walsh (2010) Drug Discov Today 15: 773-780. In certain cases PA-rich sequences can also comprise Ser as a minor constituent. For example, in case the linker consists of proline and alanine, serine can also be considered as minor constituent.

Generally, it is preferred herein that these “minor” amino acids (other than proline, alanine and, optionally, serine) are not present in the linker as described herein. In accordance with the above, the amino acid sequence of the linker (the linker) may, in particular, consist exclusively of proline, alanine and, optionally, serine residues (i.e. no other amino acid residues are present in the amino acid sequence of the linker (the linker)).

The herein provided protein can comprise an N-terminal signal polypeptide sequence, for example, the N-terminal signal polypeptide sequence of PDGFR, particularly of human PDGFRα. The N-terminal signal polypeptide sequence can have an amino acid sequence as shown in SEQ ID No. 10 or said N-terminal signal polypeptide sequence can be a polypeptide encoded by a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID No. 9.

The protein can further comprise a tag, e.g. a purification tag, such as a His-tag. Herein also other established tags can be used, like HA-tag, Flag-tag, c-myc-tag, V5-tag or C9-tag. These tags can be used in the place of an His-tag or in addition thereto. The tags can be used in the purification and detection of the herein provided protein. By using antibodies specifically binding to the tag (e.g. via ELISA assays, like chemiluminescence ELISA (CLIA) and AlphaLISA), e.g. the level of protein can be reliably and rapidly assessed and/or purification be facilitated.

The purification tag can have an amino acid sequence as shown in SEQ ID No. 12 or it can be a polypeptide encoded by a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID No. 11.

Primarily, the term “tag” refers to a “protein tag”. The terms “tag” and “protein tag” are known in the art; see, inter alia, Fritze C E, Anderson T R. “Epitope tagging: general method for tracking recombinant proteins”. Methods Enzymol. 2000; 327: 3-16; Brizzard B, Chubet R. Epitope tagging of recombinant proteins. Curr Protoc Neurosci. 2001 May; Chapter 5: Unit 5.8; and/or Terpe K. Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol. 2003 January; 60(5):523-33.

Typically, the tag to be used herein is a protein tag that is fused to the protein. For example, a nucleic acid encoding the tag can be fused to a nucleic acid encoding a protein comrpsing a PDGFR domain and a VEGFR domain, so that a fusion protein comprising both the tag and the PDGFR domain and a VEGFR domain is expressed. The tag(s) can be fused to the 5′-end of the nucleic acid encoding PDGFR domain and a VEGFR domain, inserted within the nucleic acid and/or fused to the 3′-end of the nucleic acid encoding PDGFR domain and a VEGFR domain. Thus, the resulting fusion protein can comprise (a) tag(s) at the N-terminus, internally (i.e. within the PDGFR domain and VEGFR domain), and/or at the C-terminus.

Various tags are known in the art and can be used in accordance with the present invention. Usually, a tag to be used herein has a low molecular weight of about 1-3 kDa, preferably of about 1 kDa. Exemplary, non-limiting low molecular weight tags are HA-tag, His-tag, Flag-tag, c-myc-tag, V5-tag or C9-tag. The Flag-tag to be used herein can be 1×Flag-tag or 3×Flag-tag. The low molecular weight is reflected in the length of the tag, i.e. the number of amino acid residues of which the tag consists. For example, His-tag (6 amino acids), HA-tag (9 amino acids), FLAG-tag (8 amino acids), or 3×FLAG-tag (22 amino acids) can be used herein.

The domains can be arranged in any order from N-terminus to C-terminus. Preferably, the protein is arranged from N-terminus to C-terminus in the order:

(optional signal sequence)-one or more domains of PDGFR-(optional linker)-one or more domains of VEGFR-(optional purification tag);

(optional signal sequence)-one or more domains of VEGFR-(optional linker)-one or more domains of PDGFR-(optional purification tag);

(optional signal sequence)-(optional linker)-one or more domains of VEGFR-one or more domains of PDGFR-(optional purification tag);

(optional signal sequence)-(optional linker)-one or more domains of PDGFR-one or more domains of VEGFR-(optional purification tag);

(optional signal sequence)-(optional linker)-one or more domains of PDGFR-(optional linker)-one or more domains of VEGFR-(optional linker)-(optional purification tag).

The domains can be arranged in any order from N-terminus to C-terminus. More preferably, the protein is arranged from N-terminus to C-terminus in the order:

(optional signal sequence)-one or more domains of PDGFR-(PAS/PA)-one or more domains of VEGFR-(optional purification tag);

(optional signal sequence)-one or more domains of VEGFR-(PAS/PA)-one or more domains of PDGFR-(optional purification tag);

(optional signal sequence)-(PAS/PA)-one or more domains of VEGFR-one or more domains of PDGFR-(optional purification tag);

(optional signal sequence)-(PAS/PA)-one or more domains of PDGFR-one or more domains of VEGFR-(optional purification tag);

(optional signal sequence)-(PAS/PA)-one or more domains of PDGFR-(PAS/PA)-one or more domains of VEGFR-(PAS/PA)-(optional purification tag).

The domains can be arranged in any order from N-terminus to C-terminus. Further preferably, the protein is arranged from N-terminus to C-terminus in the order:

(optional signal sequence)-one or more domains of PDGFR-(GGGGS (SEQ ID NO: 73))n-(PAS/PA) -(GGGGS (SEQ ID NO: 73))n-one or more domains of VEGFR-(optional purification tag), wherein, n=0-5, e.g. 1, 2, 3, 4 or 5 and preferably 3;

(optional signal sequence)-one or more domains of VEGFR-(GGGGS (SEQ ID NO: 73))n-(PAS/PA) -(GGGGS (SEQ ID NO: 73))n-one or more domains of PDGFR-(optional purification tag);

wherein, n=0-5, e.g. 1, 2, 3, 4 or 5 and preferably 3.

In a preferred embodiment, a protein is provided herein, wherein said protein comprises

-   (a) a protein having an amino acid sequence as shown in SEQ ID No.     14, SEQ ID No. 22, SEQ ID No. 24, SEQ ID No. 26, SEQ ID No. 28, SEQ     ID No. 30, SEQ ID No. 32, SEQ ID No. 34, SEQ ID No. 36, SEQ ID No.     38, SEQ ID No. 40, SEQ ID No. 42 or SEQ ID No. 44; -   (b) a protein as defined in (a) wherein 1 to 10 amino acids are     deleted, inserted, added or substituted; -   (c) a polypeptide encoded by a nucleic acid molecule having a     nucleotide sequence as shown in SEQ ID No. 13, SEQ ID No. 21, SEQ ID     No. 23, SEQ ID No. 25, SEQ ID No. 27, SEQ ID No. 29, SEQ ID No. 31,     SEQ ID No. 33, SEQ ID No. 35, SEQ ID No. 37, SEQ ID No. 39, SEQ ID     No. 41 or SEQ ID No. 43; -   (d) a polypeptide having an amino acid sequence encoded by a nucleic     acid hybridizing under stringent conditions to the complementary     strand of nucleic acid molecules as defined in (c); -   (e) a polypeptide having at least 70% identity to the polypeptide of     any one of (a) to (d); and -   (f) a polypeptide having an amino acid sequence encoded by a nucleic     acid being degenerate as a result of the genetic code to the     nucleotide sequence of a nucleic acid as defined in (c) or (d).

The following relates to proteins (or a functional fragment or derivative thereof) to be used in accordance with the present invention.

The meaning of the term “protein” and “nucleic acid sequence(s)/molecule(s)” are well known in the art and are used accordingly in context of the present invention.

For example, the term “protein” as used herein refers to a biomolecule consisting of one or more chains of amino acid residues. The terms “polypeptide” and “chain of amino acid residues” can be used interchangeably herein. A single linear chain of amino acid residues is usually called a polypeptide. The term protein usually refers to a biological molecule in a stable conformation (i.e. implies that a three-dimensional structure has formed). Normally, a protein contains more than 20-30 amino acid residues, particularly more than 50 amino acid residues. A protein can contain up to 3000 amino acid residues, e.g. up to 1500 amino acid residues. Yet, even larger proteins are envisaged herein.

The individual amino acid residues are bonded together by peptide bonds. In general, the genetic code specifies 20 standard amino acids; however, also the use of non-standard amino acids like selenocysteine is envisaged herein. Also chemical modification e.g. post-translational modification is envisaged herein.

Short proteins can also be synthesized chemically by a family of methods known as peptide synthesis, which rely on organic synthesis techniques such as chemical ligation.

As described herein, a method for the preparation of the herein disclosed protein is provided. The method can comprise culturing the host cell as provided herein and isolating said protein from the culture or from the (host) cell(s). As described herein, a fusion protein as provided herein can be prepared by expressing the nucleic acid molecule as provided herein, and optionally, by isolating the expressed fusion protein.

Alternatively, the protein can be prepared by culturing/raising the host comprising the nucleotide sequence encoding the linker, particularly the linker consisting of proline, alanine and, optionally serine. Thus, the linker can be expressed in the host and/or optionally, isolated. The linker consisting of proline, alanine and, optionally, serine can then be conjugated to the PDGFR and/or VEGFR domain, e.g., via a peptide bond or a non-peptide bond. In particular, the PDGFR or VEGFR domain can be site-specifically conjugated, e.g., in the presence of an activating agent such as N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) or as an N-hydroxysuccinimide (NHS) ester (Hermanson (1996) Bioconjugate Techniques, 1st edition, Academic Press, San Diego, Calif.) to the N-terminus of the linker, particularly the linker consisting of proline, alanine and, optionally, serine. Alternatively, the PDGFR or VEGFR domain can be site-specifically conjugated to the C-terminus of the linker, particularly the linker consisting of proline, alanine and, optionally, serine, e.g., in the presence of an activating agent such as EDC or after activation as an NHS ester.

It is preferred herein that the protein is a fusion protein.

In order to prepare a fusion protein, a nucleotide sequence encoding the PDGFR domain, can be operably linked in the same reading frame to the VEGFR domain. If the fusion protein comprises a linker (particularly a linker consisting of proline, alanine and, optionally, serine), the fusion protein can, for example, be prepared in that a nucleotide sequence encoding the PDGFR domain, is operably linked in the same reading frame to a nucleotide sequence encoding the linker, and the nucleotide sequence encoding the linker is operably linked in the same reading frame to a nucleotide sequence encoding the VEGFR domain.

Thus, a nucleic acid molecule provided herein can encode a fusion protein/heterologous drug conjugate comprising a PDGFR domain, a linker consisting of proline, alanine and, optionally, serine, and a VEGFR domain.

As used herein, the term “operably linked” refers to a juxtaposition, wherein the components in question are in a relationship permitting them to both function in their intended manner.

The nucleotide sequence encoding the linker, particularly the linker consisting of proline, alanine and, optionally, serine can be conjugated to the nucleotide sequence encoding the PDGFR domain and/or VEGFR domain seamlessly, i.e., no further spacers intersperse these sequences. Spacers can cause an immune response in the subject receiving the fusion protein that carries such a spacer. Therefore, the nucleotide sequence encoding the linker can be conjugated to the nucleotide sequence encoding the PDGFR domain and/or VEGFR domain seamlessly. As used herein, “seamless” means that the nucleotide sequence encoding the linker is directly conjugated to the nucleotide sequence encoding the PDGFR domain and/or VEGFR domain. Thus, no additional nucleotides are introduced that encode amino acid residues other than proline, alanine and, optionally, serine.

Alternatively, a spacer structure can be comprised between the linker and the PDGFR domain and/or VEGFR domain. Thus, in certain aspects of the invention, a nucleotide sequence encoding an amino acid spacer is inserted between the nucleotide sequence encoding the linker and the nucleotide sequence encoding PDGFR domain and/or VEGFR domain. An exemplary spacer can be a protease sensitive cleavage site, a serine/glycine-linker, an affinity tag such as the His6-tag or the Strep-tag II, a signal peptide, retention peptide, a targeting peptide like a membrane translocation peptide or additional effector domains, e.g., antibody fragments for tumour targeting associated with an anti-tumour toxin or an enzyme for prodrug activation etc. The protein comprising a spacer can have a plasma protease cleavage site that allows the controlled release of said protein. Spacers of different types or lengths may be identified without undue burden to obtain/preserve optimal biological activity of the proteins. An exemplary serine/glycine-linker can have the sequence (GGGGS (SEQ ID NO: 73))n, wherein, n=0-5, e.g. 1, 2, 3, 4 or 5. Preferably, n=3. If n=0 the serine/glycine-linker is absent. For example, the serine/glycine-linker may be arranged in the protein in the following order:

(optional signal sequence)-one or more domains of PDGFR-(GGGGS (SEQ ID NO: 73))n-(PAS/PA) -(GGGGS (SEQ ID NO: 73))n-one or more domains of VEGFR-(optional purification tag), wherein, n=0-5, e.g. 1, 2, 3, 4 or 5 and preferably 3; or

(optional signal sequence)-one or more domains of VEGFR-(GGGGS (SEQ ID NO: 73))n-(PAS/PA) -(GGGGS (SEQ ID NO: 73))n-one or more domains of PDGFR-(optional purification tag);

wherein, n=0-5, e.g. 1, 2, 3, 4 or 5 and preferably 3.

Nucleic acid sequences with a certain level of identity to the herein provided sequences can be identified by the skilled person using methods known in the art, e.g. by using hybridization assays or by using alignments, either manually or by using computer programs such as those mentioned herein below in connection with the definition of the term “hybridization” and degrees of homology.

The nucleic acid sequence may be at least 70% identical to the nucleic acid sequence as shown in any one of SEQ ID No. 3, 5, 7, 13, 15, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65 or 67.

More preferably, the nucleic acid sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% identical to the nucleic acid sequence as shown in any one of SEQ ID No. 3, 5, 7, 13, 15, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65 or 67, wherein the higher values are preferred. Most preferably, the nucleic acid sequence is at least 99% identical to the nucleic acid sequence as shown in any one of SEQ ID No. 3, 5, 7, 13, 15, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65 or 67.

Hybridization assays for the characterization of nucleic acids with a certain level of identity to the nucleic acid sequences as provided herein are well known in the art; see e.g. Sambrook, Russell “Molecular Cloning, A Laboratory Manual”, Cold Spring Harbor Laboratory, N.Y. (2001); Ausubel, “Current Protocols in Molecular Biology”, Green Publishing Associates and Wiley Interscience, N.Y. (1989). The term “hybridization” or “hybridizes” as used herein may relate to hybridizations under stringent or non-stringent conditions. If not further specified, the conditions are preferably non-stringent. Said hybridization conditions may be established according to conventional protocols described, e.g., in Sambrook (2001) loc. cit.; Ausubel (1989) loc. cit., or Higgins and Hames (Eds.) “Nucleic acid hybridization, a practical approach” IRL Press Oxford, Washington D.C., (1985). The setting of conditions is well within the skill of the artisan and can be determined according to protocols described in the art. Thus, the detection of only specifically hybridizing sequences will usually require stringent hybridization and washing conditions such as, for example, the highly stringent hybridization conditions of 0.1×SSC, 0.1% SDS at 65° C. or 2×SSC, 60° C., 0.1% SDS. Low stringent hybridization conditions for the detection of homologous or not exactly complementary sequences may, for example, be set at 6×SSC, 1% SDS at 65° C. As is well known, the length of the probe and the composition of the nucleic acid to be determined constitute further parameters of the hybridization conditions. It is envisaged herein that a nucleic acid can be a primer or probe, for example, a nucleic acid hybridizing under stringent conditions to the complementary strand of the nucleic acid of the herein provide protein (or of a fragment thereof as defined herein) the like as defined and provided herein above. Primers and probes are often in the range of 10-30 nucleotides. Thus, herein provided is a nucleic acid (like a primer or probe) hybridizing under stringent conditions to the complementary strand of the protein as defined and provided herein above, wherein said hybridizing nucleic acid is smaller than 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, or 20 nucleotides and is larger than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides. Preferably, the nucleic acid has a length of 10 to 35 nucleotides, more preferably 15 to 25 nucleotides, particularly preferred a length of 18 to 21, e.g. 18, 19, 20 or 21 nucleotides.

In accordance with the present invention, the terms “homology” or “percent homology” or “identical” or “percent identity” or “percentage identity” or “sequence identity” in the context of two or more nucleic acid sequences refers to two or more sequences or subsequences that are the same, or that have a specified percentage of nucleotides that are the same (at least 70%, 75%, 80%, 85%, most preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% identity, most preferably at least 99% identity), when compared and aligned for maximum correspondence over a window of comparison (preferably over the full length), or over a designated region as measured using a sequence comparison algorithm as known in the art, or by manual alignment and visual inspection. Sequences having, for example, 75% to 90% or greater sequence identity may be considered to be substantially identical. Such a definition also applies to the complement of a test sequence. Preferably the described identity exists over a region that is at least about 15 to 25 nucleotides in length, more preferably, over a region that is at least about 50 to 100 nucleotides in length and most preferably over the full length. Those having skill in the art will know how to determine percent identity between/among sequences using, for example, algorithms such as those based on CLUSTALW computer program (Thompson Nucl. Acids Res. 2 (1994), 4673-4680) or FASTDB (Brutlag Comp. App. Biosci. 6 (1990), 237-245), as known in the art.

Although the FASTDB algorithm typically does not consider internal non-matching deletions or additions in sequences, i.e., gaps, in its calculation, this can be corrected manually to avoid an overestimation of the % identity. CLUSTALW, however, does take sequence gaps into account in its identity calculations. Also available to those having skill in this art are the BLAST and BLAST 2.0 algorithms (Altschul, (1997) Nucl. Acids Res. 25:3389-3402; Altschul (1993) J. Mol. Evol. 36:290-300; Altschul (1990) J. Mol. Biol. 215:403-410). The BLASTN program for nucleic acid sequences uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=4, and a comparison of both strands. The BLOSUM62 scoring matrix (Henikoff (1989) PNAS 89:10915) uses alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.

In order to determine whether a nucleotide residue in a nucleic acid sequence corresponds to a certain position in the nucleotide sequence of e.g. SEQ ID No. 3, 5, 7, 13 or 15, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65 or 67, respectively, the skilled person can use means and methods well-known in the art, e.g., alignments, either manually or by using computer programs such as those mentioned herein. For example, BLAST 2.0, which stands for Basic Local Alignment Search Tool BLAST (Altschul (1997), loc. cit.; Altschul (1993), loc. cit.; Altschul (1990), loc. cit.), can be used to search for local sequence alignments. BLAST, as discussed above, produces alignments of nucleotide sequences to determine sequence similarity. Because of the local nature of the alignments, BLAST is especially useful in determining exact matches or in identifying similar sequences. The fundamental unit of BLAST algorithm output is the High-scoring Segment Pair (HSP). An HSP consists of two sequence fragments of arbitrary but equal lengths whose alignment is locally maximal and for which the alignment score meets or exceeds a threshold or cut-off score set by the user. The BLAST approach is to look for HSPs between a query sequence and a database sequence, to evaluate the statistical significance of any matches found, and to report only those matches which satisfy the user-selected threshold of significance. The parameter E establishes the statistically significant threshold for reporting database sequence matches. E is interpreted as the upper bound of the expected frequency of chance occurrence of an HSP (or set of HSPs) within the context of the entire database search. Any database sequence whose match satisfies E is reported in the program output.

Analogous computer techniques using BLAST (Altschul (1997), loc. cit.; Altschul (1993), loc. cit.; Altschul (1990), loc. cit.) are used to search for identical or related molecules in nucleotide databases such as GenBank or EMBL. This analysis is much faster than multiple membrane-based hybridizations. In addition, the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or similar. The basis of the search is the product score, which is defined as:

$\frac{\%{sequence}{identity} \times \%{maximum}{BLAST}{score}}{100}$ and it takes into account both the degree of similarity between two sequences and the length of the sequence match. For example, with a product score of 40, the match will be exact within a 1-2% error; and at 70, the match will be exact. Similar molecules are usually identified by selecting those, which show product scores between 15 and 40, although lower scores may identify related molecules. Another example for a program capable of generating sequence alignments is the CLUSTALW computer program (Thompson (1994) Nucl. Acids Res. 2:4673-4680) or FASTDB (Brutlag (1990) Comp. App. Biosci. 6:237-245), as known in the art.

The explanations and definitions given herein above in respect of “homology/identity of nucleic acid sequences” apply, mutatis mutandis, to “amino acid sequences” of the herein provided proteins as depicted in any one of SEQ ID No.s: 4, 6, 8, 14, 16, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66 or 68, respectively, as explained below.

The proteins to be used in accordance with the present invention may have at least 70% identity/similarity to the proteins having the amino acid sequence as shown in any one of SEQ ID No.s: 4, 6, 8, 14, 16, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66 or 68, respectively. More preferably, the proteins has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% identity/similarity to the proteins having the amino acid sequence as shown in any one of SEQ ID No.s: 4, 6, 8, 14 and 16, respectively, wherein the higher values are preferred. Most preferably, the proteins have at least 99% identity/similarity to the proteins having the amino acid sequence as shown in any one of SEQ ID No.s: 4, 6, 8, 14, 16, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66 or 68, respectively.

Also (a) (functional) fragment(s) or (a) (functional) derivative(s) of the herein provided proteins can be used, for example, (functional) fragment(s) or (functional) derivative(s) of the proteins having the amino acid sequence as shown in any one of SEQ ID No.s: 4, 6, 8, 14 and 16, respectively.

Thus, a (functional) fragment of the protein(s) provided herein and to be used in accordance with the present invention can be any of the above specific proteins as shown in any one of SEQ ID No.s: 4, 6, 8, 14, 16, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66 or 68, respectively, wherein one or more amino acids are deleted.

The term “one or more amino acids” refers for example to “1, 2, 3, 4, 5, 6, 7, 8, 9 or 10” amino acids.

A (functional) derivative(s) of the protein(s) provided herein and to be used in accordance with the present invention can be any of the above specific proteins as shown in any one of SEQ ID No.s: 4, 6, 8, 14, 16, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66 or 68, respectively, wherein one or more amino acids are inserted, added or substituted.

A (functional) fragment of the proteins provided herein and to be used in accordance with the present invention can consist of at least 100, 120, 140, 160, or 180 contiguous amino acids of the amino acid sequence as shown in any one of SEQ ID No.s: 4, 6, 8, 14, 16, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66 or 68.

The fragment or derivative preferably has the same (or essentially the same) biological activity as the full length protein from which it is derived, the full length polypeptide having the amino acid sequence as shown in as shown in any one of SEQ ID No.s: 4, 6, 8, 14, 16, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66 or 68, respectively. In this sense, the fragment or derivative is a “functional” fragment or derivative to be used herein.

The herein provided protein (having the amino acid sequence as shown in as shown in any one of SEQ ID No.s: 4, 6, 8, 14, 16, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66 or 68, may have one or more amino acids deleted, inserted, added and/or substituted provided that the polypeptide maintains essentially the biological activity which is characteristic of the polypeptides from which it is derived.

Preferably, any such deletions, insertions, additions and/or substitutions (in this context particularly substitutions) are conservative, i.e. amino acids are substituted by amino acids having the same or similar characteristics. For example, a hydrophobic amino acid will preferably be substituted by another hydrophobic amino acid and so on.

The “biological activity” characteristic of the herein provided proteins can, for example, be considered as capacity to bind the ligand (PDGF and VEGF, respectively) as defined herein. As regards the linker, particularly the linker consisting of proline, alanine and, optionally, serine, the “biological activity” can particularly be considered as capacity to form random conformation.

Herein provided is a nucleic acid molecule encoding the herein provided protein.

For example, “nucleic acid sequence(s)/molecule(s)” as used herein refer(s) to all forms of naturally occurring or recombinantly generated types of nucleic acids and/or nucleic acid sequences/molecules as well as to chemically synthesized nucleic acid sequences/molecules. This term also encompasses nucleic acid analogs and nucleic acid derivatives such as e.g. locked DNA, PNA, oligonucleotide thiophosphates and substituted ribo-oligonucleotides. Furthermore, the term “nucleic acid sequence(s)/molecules(s)” also refers to any molecule that comprises nucleotides or nucleotide analogs. The terms “nucleic acid” and “nucleic acid molecule” are used interchangeably herein.

Preferably, the term “nucleic acid sequence(s)/molecule(s)” refers to deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The “nucleic acid sequence(s)/molecule(s)” may be made by synthetic chemical methodology known to one of ordinary skill in the art, or by the use of recombinant technology, or may be isolated from natural sources, or by a combination thereof. The DNA and RNA may optionally comprise unnatural nucleotides and may be single or double stranded. “Nucleic acid sequence(s)/molecule(s)” also refers to sense and anti-sense DNA and RNA, that is, a nucleotide sequence which is complementary to a specific sequence of nucleotides in DNA and/or RNA.

Furthermore, the term “nucleic acid sequence(s)/molecule(s)” may refer to DNA or RNA or hybrids thereof or any modification thereof that is known in the state of the art (see, e.g., U.S. Pat. Nos. 5,525,711, 4,711,955, 5,792,608 or EP 302175 for examples of modifications). The nucleic acid molecule(s) may be single- or double-stranded, linear or circular, natural or synthetic, and without any size limitation. For instance, the nucleic acid molecule(s) may be genomic DNA, cDNA, mRNA, antisense RNA, ribozymal or a DNA encoding such RNAs or chimeroplasts (Colestrauss, Science (1996), 1386-1389). Said nucleic acid molecule(s) may be in the form of a plasmid or of viral DNA or RNA. “Nucleic acid sequence(s)/molecule(s)” may also refer to (an) oligonucleotide(s), wherein any of the state of the art modifications such as phosphothioates or peptide nucleic acids (PNA) are included.

Further, a vector comprising the nucleic acid is provided.

Many suitable vectors are known to those skilled in molecular biology. The choice of a suitable vector depends on the function desired, including plasmids, cosmids, viruses, bacteriophages and other vectors used conventionally in genetic engineering.

Preferably, the vector is a plasmid, more preferably a plasmid based on the generic E. coli expression vector pASK37, pASK75 or pXL2.

Methods which are well known to those skilled in the art can be used to construct various plasmids; see, for example, the techniques described in Sambrook (2001) loc cit. and Ausubel (1989) loc. cit. Typical plasmid vectors include, e.g., pQE-12, the pUCseries of plasmids, pBluescript (Stratagene), the pET series of expression vectors (Novagen) or pCRTOPO (Invitrogen), lambda gt11, pJOE, the pBBR1-MCS series, pJB861, pBSMuL, pBC2, pUCPKS, pTACT1. Typical vectors compatible with expression in mammalian cells inlcude E-027 pCAG Kosak-Cherry (L45a) vector system, pREP (Invitrogen), pCEP4 (Invitrogen), pMC1neo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2neo, pBPV-1, pdBPVMMTneo, pRSVgpt, pRSVneo, pSV2-dhfr, pIZD35, Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pRc/CMV, pcDNA1, pcDNA3 (Invitrogen), pcDNA3.1, pSPORT1 (GIBCO BRL), pGEMHE (Promega), pLXIN, pSIR (Clontech), pIRES-EGFP (Clontech), pEAK-10 (Edge Biosystems) pTriEx-Hygro (Novagen) and pCINeo (Promega). Non-limiting examples for plasmid vectors suitable for Pichia pastoris comprise e.g. the plasmids pAO815, pPIC9K and pPIC3.5K (all Invitrogen).

Generally, vectors can contain one or more origins of replication (ori) and inheritance systems for cloning or expression, one or more markers for selection in the host, e.g., antibiotic resistance, and one or more expression cassettes. Examples of suitable origins of replication include, for example, the full length ColE1, its truncated versions such as those present on the pUC plasmids, the SV40 viral and the M13 phage origins of replication. Non-limiting examples of selectable markers include ampicillin, chloramphenicol, tetracycline, kanamycin, dhfr, gpt, neomycin, hygromycin, blasticidin or geneticin.

Further, said vector comprises a regulatory sequence that is operably linked to said nucleotide sequence or the nucleic acid molecule defined herein.

The coding sequence(s), e.g., said nucleotide sequence encoding the herein provided protein comprising a PDGFR domain and a VEGFR domain, and preferably, a linker consisting of PAS or PA, comprised in the vector can be linked to (a) transcriptional regulatory element(s) and/or to other amino acid encoding sequences using established methods. Such regulatory sequences are well known to those skilled in the art and include, without being limiting, regulatory sequences ensuring the initiation of transcription, internal ribosomal entry sites (IRES) and, optionally, regulatory elements ensuring termination of transcription and stabilization of the transcript. Non-limiting examples for such regulatory sequences ensuring the initiation of transcription comprise promoters, a translation initiation codon, enhancers, insulators and/or regulatory elements ensuring transcription termination. Further examples include Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing, nucleic acid sequences encoding secretion signals or, depending on the expression system used, signal polypeptide sequences capable of directing the expressed protein to a cellular compartment or to the culture medium.

Examples of suitable promoters include, without being limiting, the cytomegalovirus (CMV) promoter, SV40 promoter, RSV (Rous sarcome virus) promoter, the lacZ promoter, chicken β-actin promoter, CAG promoter (a combination of chicken β-actin promoter and cytomegalovirus immediate-early enhancer), human elongation factor 1a promoter, AOX1 promoter, GAL1 promoter, CaM-kinase promoter, the lac, trp or tac promoter, the lacUV5 promoter, the T7 or T5 promoter, the Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) polyhedral promoter or a globin intron in mammalian and other animal cells. One example of an enhancer is, e.g., the SV40 enhancer. Non-limiting additional examples for regulatory elements/sequences ensuring transcription termination include the SV40 poly-A site, the tk poly-A site or the AcMNPV polyhedral polyadenylation signals.

Furthermore, depending on the expression system, leader sequences capable of directing the polypeptide to a cellular compartment or secreting it into the medium may be added to the coding sequence of the nucleic acid molecule provided herein. The leader sequence(s) is (are) assembled in frame with translation, initiation and termination sequences, and preferably, a leader sequence is capable of directing secretion of translated protein, or a portion thereof, into the periplasmic space or into the extracellular medium. Suitable leader sequences are, for example, the signal polypeptide sequences of BAP (bacterial alkaline phosphatase), CTB (cholera toxin subunit B), DsbA, ENX, OmpA, PhoA, stII, OmpT, PelB, Tat (Twin-arginine translocation) in E. coli, and the signal polypeptide sequences of bovine growth hormone, human chymotrypsinogen, human factor VIII, human ig-kappa, human insulin, human interleukin-2, luciferase from Metrida or Vargula, human trypsinogen-2, inulinase from Kluyveromyces marxianus, mating factor alpha-1 from Saccharomyces cerevisiae, mellitin, human azurocidin and the like in eukaryotic cells.

The vectors may also contain an additional expressible nucleic acid sequence coding for one or more chaperones to facilitate correct protein folding.

The vector present in the host of the described herein can either be an expression vector, or the vector can mediate the stable integration of the nucleic acid molecule as provided herein into the genome of the host cell in such a manner that expression of the protein is ensured. Means and methods for selecting a host cell in which the nucleic acid molecule as provided herein has been successfully introduced such that expression of the protein is ensured are well known in the art and have been described (Browne (2007) Trends Biotechnol. 25:425-432; Matasci (2008) Drug Discov. Today: Technol. 5:e37-e42; Wurm (2004) Nat. Biotechnol. 22:1393-1398).

Preferably, the vector to be used herein is an expression vector. An expression vector to be used herein is capable of directing the replication and the expression of the nucleic acid molecule provided herein, e.g., the nucleic acid molecule comprising the nucleotide sequence encoding the protein provided herein.

Herein disclosed is (a) host cell (s) comprising the herein provided nucleic acid or the herein provided vector. The host cell can be a eukaryotic host cell or a prokaryotic host cell. A preferred prokaryotic host cell is E. coli. The eukaryotic host cell can be a fungal or animal cell. Preferred animal cell(s) is(are) (a) HEK cell(s) or (a) CHO cell(s).

The present disclosure also relates to (a) host cell(s) or a non-human host transformed with a vector or the nucleic acid molecule as provided herein. It will be appreciated that the term “host cell or a non-human host transformed with the vector”, in accordance with the present disclosure, relates to a host cell or a non-human host that comprises the vector or the nucleic acid molecule as provided herein.

Host cells for the expression of polypeptides are well known in the art and comprise prokaryotic cells as well as eukaryotic cells. Thus, the host/host cell can be selected from the group consisting of a bacterium, a mammalian cell, an algal cell, a ciliate, yeast and a plant cell.

Suitable bacterial expression hosts comprise, e.g., strains derived from Escherichia coli JM83, W3110, KS272, TG1, BL21 (such as BL21(DE3), BL21(DE3)PlysS, BL21(DE3)RIL, BL21(DE3)PRARE), Origami (K-12), Origami B or Rosetta. For vector modification, PCR amplification and ligation techniques, see methods described in Sambrook (2001) loc. cit.

Additionally, baculoviral systems can also be used as a vector in order to express the nucleic acid molecules of the invention in eukaryotic expression systems. In these aspects, the pFBDM vector can be used as an expression vector. The insertion into the MultiBac baculoviral DNA is mediated via the Tn7 transposition sequence upon transformation of DH10 MultiBac E. coli cells (Berger (2013) J. Vis. Exp. 77:50159, Fitzgerald (2006) Nat. Methods. 2006 3:1021-1032.). Virus amplification and expression can be performed in Sf21 (Spodoptera frugiperda) or High Five (Trichoplusia ni) cells.

The nucleic acid molecules and/or vectors as described herein above may be designed for introduction into cells by, e.g., non-chemical methods (electroporation, sonoporation, optical transfection, gene electrotransfer, hydrodynamic delivery or naturally occurring transformation upon contacting cells with the nucleic acid molecule of the invention), chemical-based methods (calcium phosphate, DMSO, PEG, liposomes, DEAE-dextrane, polyethylenimine, nucleofection etc.), particle-based methods (gene gun, magnetofection, impalefection), phage or phagemid vector-based methods and viral methods. For example, expression vectors derived from viruses such as retroviruses, vaccinia virus, adeno-associated virus, herpes viruses, Semliki Forest Virus or bovine papilloma virus, may be used for delivery of the nucleic acid molecules into a targeted cell population.

Preferably, the nucleic acid molecules and/or vectors provided herein are designed for transformation of electrocompetent E. coli by electroporation or for stable transfection of CHO cells by calcium phosphate, polyethylenimine or lipofectaminetransfection (Pham (2006) Mol. Biotechnol. 34:225-237; Geisse (2012) Methods Mol. Biol. 899:203-219; Hacker (2013) Protein Expr. Purif. 92:67-76).

Typical bacteria include Escherichia, Corynebacterium (glutamicum), Pseudomonas (fluorescens), Lactobacillus, Streptomyces, Salmonella Bacillus (such as Bacillus megaterium or Bacillus subtilis), or Corynebacterium (like Corynebacterium glutamicum). The most preferred bacterium host herein is E. coli. An exemplary ciliate to be used herein is Tetrahymena, e.g. Tetrahymena thermophila.

Typical mammalian cells include, Hela, HEK293, HEK293T, H9, Per.C6 and Jurkat cells, mouse NIH3T3, NS0 and C127 cells, COS 1, COS 7 and CV1, quail QC1-3 cells, mouse L cells, mouse sarcoma cells, Bowes melanoma cells and Chinese hamster ovary (CHO) cells. Most preferred mammalian host cells in accordance with the present invention are CHO cells. An exemplary host to be used herein is Cricetulus, e.g. Cricetulus griseus (Chinese hamster). Also, human embryonic kidney (HEK) cells are preferred.

Other suitable eukaryotic host cells are e.g. yeasts such as Pichia pastoris, Kluyveromyces lactis, Saccharomyces cerevisiae and Schizosaccharomyces pombe or chicken cells, such as e.g. DT40 cells. Insect cells suitable for expression are e.g. Drosophila S2, Drosophila Kc, Spodoptera Sf9 and Sf21 or Trichoplusia Hi5 cells. Preferable algal cells are Chlamydomonas reinhardtii or Synechococcus elongatus cells and the like. An exemplary plant is Physcomitrella, for example Physcomitrella patens. An exemplary plant cell is a Physcomitrella plant cell, e.g. a Physcomitrella patens plant cell.

Also within the scope of the present invention are primary mammalian cells or cell lines. Primary cells are cells which are directly obtained from an organism. Suitable primary cells are, for example, mouse embryonic fibroblasts (MEF), mouse primary hepatocytes, cardiomyocytes and neuronal cells as well as mouse muscle stem cells (satellite cells), human dermal and pulmonary fibroblasts, human epithelial cells (nasal, tracheal, renal, placental, intestinal, bronchial epithelial cells), human secretory cells (from salivary, sebaceous and sweat glands), human endocrine cells (thyroid cells), human adipose cells, human smooth muscle cells, human skeletal muscle cells, human leucocytes such as B-cells, T-cells, NK-cells or dendritic cells and stable, immortalized cell lines derived thereof (for example hTERT or oncogene immortalized cells). Appropriate culture media and conditions for the above described host cells are known in the art.

The host cells may e.g. be employed to produce large amounts of the nucleic acid molecule provided herein, and/or the protein as provided herein. Accordingly, herein provided is a method for preparing the nucleic acid molecule or the vector provided herein, the method comprising culturing the host or host cell of the invention under suitable conditions and optionally isolating the produced nucleic acid molecule and/or vector. Furthermore, herein provided is to a method for preparing the protein as described and provided herein, the method comprising culturing the host or host cell as provided herein under suitable conditions and optionally isolating the produced protein. Particularly in this aspect, it is envisaged that the protein is a fusion protein.

Alternatively, the method can also comprise culturing the host or host cell as provided herein (i.e. a host or host cell comprising a nucleic acid encoding a linker, particularly a linker consisting of proline, alanine and, optionally, serine, as provided herein) and/or culturing the host or host cell comprising a nucleic acid encoding the PDGFR domain and/or VEGFR domain as defined herein, and optionally isolating the produced linker and/or isolating the produced PDGFR domain and/or VEGFR domain, and further optionally conjugating the linker and the PDGFR domain and/or VEGFR domain (e.g. by chemical coupling) to produce the protein.

“Culturing the host or host cell” includes in this context expression of the linker as defined herein and/or of the PDGFR domain and/or of the VEGFR domain in the host or host cell.

Suitable conditions for culturing prokaryotic or eukaryotic host cells are well known to the person skilled in the art. For example, bacteria such as e.g. E. coli can be cultured under aeration in Luria Bertani (LB) medium, typically at a temperature from 4 to about 37° C. To increase the yield and the solubility of the expression product, the medium can be buffered or supplemented with suitable additives known to enhance or facilitate both. In those cases where an inducible promoter controls the nucleic acid molecule of the invention in the vector present in the host cell, expression of the polypeptide can be induced by addition of an appropriate inducing agent, such as, e.g., isopropyl-β-D-thiogalactopyranoside (IPTG) or anhydrotetracycline (aTc) as employed in the appended examples. Suitable expression protocols and strategies have been described in the art, e.g. in Sambrook (2001) loc. cit., (Gebauer (2012) Meth. Enzymol. 503:157-188) and can be adapted to the needs of the specific host cells and the requirements of the protein to be expressed, if required.

Depending on the cell type and its specific requirements, mammalian cell culture can, e.g., be carried out in RPMI, Williams' E or medium DMEM containing 10% (v/v) FCS, 2 mM L-glutamine and 100 U/ml penicillin/streptomycin. The cells can be kept, e.g., at 37° C., or at 41° C. for DT40 chicken cells, in a 5% CO2, water-saturated atmosphere. A suitable medium for insect cell culture is, e.g., TNM+10% FCS, SF900 or HyClone SFX-Insect medium. Insect cells are usually grown at 27° C. as adhesion or suspension cultures. Suitable expression protocols for eukaryotic or vertebrate cells are well known to the skilled person and can be retrieved, e.g., from Sambrook (2001) (loc. cit).

Preferably, the method for preparing the protein, nucleic acid molecule, the vector as described herein is carried out using either bacterial cells, such as, e.g., E. coli cells, or mammalian cells, such as, e.g., CHO cells. More preferably, the method is carried out using E. coli cells or CHO cells and most preferably, the method is carried out using E. coli cells.

Methods for the isolation of the encoded polypeptides produced comprise, without limitation, purification steps such as affinity chromatography (preferably using a fusion tag such as the Strep-tag II or the His6-tag), gel filtration (size exclusion chromatography), anion exchange chromatography, cation exchange chromatography, hydrophobic interaction chromatography, high pressure liquid chromatography (HPLC), reversed phase HPLC, ammonium sulfate precipitation or immunoprecipitation. These methods are well known in the art and have been generally described, e.g., in Sambrook (2001) loc. cit. Such methods provide substantially pure polypeptides. Said pure polypeptides have a homogeneity of, preferably, at least about 90 to 95% (on the protein level), more preferably, at least about 98 to 99%. Most preferably, these pure polypeptides are suitable for pharmaceutical use/applications. Depending upon the host cell/organism employed in the production procedure, the proteins provided herein of the present invention may be glycosylated or may be non-glycosylated. Preferably, the linker consisting of proline, alanine and, optionally, serine provided herein is not post-translationally modified, particularly not glycosylated. Most preferably, the linker consisting of proline, alanine and, optionally, serine provided herein is not posttranslationally modified in its side chains such as, for example, by proline hydroxylation.

In the linker that can consist of alanine, proline and, optionally, serine residues, the amino acid residues threonine or asparagine (or serine, if applicable), which is/are required for 0- or N-glycosylation, can be absent. Thus, the linker would be (essentially) devoid of post-translational modifications within the Pro/Ala/Ser or Pro/Ala sequence. This is an advantage for recombinant protein production in eukaryotic cells, like chinese hamster ovarian cells (CHO), HEK cells, or yeast, which are often chosen for the biosynthesis of complex proteins.

Herein disclosed is a composition comprising the protein as provided herein or as prepared by the method as disclosed herein above, the nucleic acid as provided herein, the vector as provided herein, or the (host) cell as provided herein.

The composition can be a pharmaceutical composition, optionally further comprising (a) pharmaceutical acceptable carrier(s).

In one aspect, the protein as provided herein or as prepared by the method as disclosed herein above, the nucleic acid as provided herein, the vector as provided herein, the cell as provided herein, or the composition as provided herein, is for use as a medicament.

In one aspect, the protein as provided herein or as prepared by the method as disclosed herein above, the nucleic acid as provided herein, the vector as provided herein, the cell as provided herein, or the composition as provided herein, is for use in therapy.

In one aspect, use of the protein as provided herein or as prepared by the method as disclosed herein above, use of the nucleic acid as provided herein, use of the vector as provided herein, use of the cell as provided herein, or use of the composition as provided herein, is disclosed for the preparation of a pharmaceutical composition for use in therapy.

In one aspect, the protein as provided herein or as prepared by the method as disclosed herein above, the nucleic acid as provided herein, the vector as provided herein, the cell as provided herein, or the composition as provided herein, can inhibit angiogenesis effectively, and is for use in the treatment of diseases associated with angiogenesis, including but not limited to all types of tumor, all types of ophthalmic disease (for example, Diabetic retinopathy (DR), Diabetic macular edema (DME), Choroidal neovascularization (CNV), Retinal vein occlusion (RVO), Central retinal vein occlusion (CRVO), Branch retinal vein occlusion (BRVO), or pathologic myopia (PM); preferably age-related macular degeneration (AMD)), cancer, renal fibrosis, cirrhosis, arthosclerosis, portal hypertension or systemic sclerosis.

Specifically, in one aspect, the protein as provided herein or as prepared by the method as disclosed herein above, the nucleic acid as provided herein, the vector as provided herein, the cell as provided herein, or the composition as provided herein, is for use in the treatment of ophthalmic disease (for example, Diabetic retinopathy (DR), Diabetic macular edema (DME), Choroidal neovascularization (CNV), Retinal vein occlusion (RVO), Central retinal vein occlusion (CRVO), Branch retinal vein occlusion (BRVO), or pathologic myopia (PM); preferably, age-related macular degeneration (AMD)), cancer, renal fibrosis, cirrhosis, arthosclerosis, portal hypertension or systemic sclerosis. In one aspect, the protein as provided herein or as prepared by the method as disclosed herein above, the nucleic acid as provided herein, the vector as provided herein, the cell as provided herein, or the composition as provided herein, is for use in inhibiting angiogenesis (particularly in a diseased patient).

Age-related macular degeneration (AMD) is the preferred ophthalmic disease to be treated herein.

In one aspect, use of the protein as provided herein or as prepared by the method as disclosed herein above, use of the nucleic acid as provided herein, use of the vector as provided herein, use of the cell as provided herein, or use of the composition as provided herein, is disclosed for the preparation of a pharmaceutical composition for the treatment of ophthalmic disease (for example, Diabetic retinopathy (DR), Diabetic macular edema (DME), Choroidal neovascularization (CNV), Retinal vein occlusion (RVO), Central retinal vein occlusion (CRVO), Branch retinal vein occlusion (BRVO), or pathologic myopia (PM); preferably age-related macular degeneration (AMD)), cancer, renal fibrosis, cirrhosis, arthosclerosis, portal hypertension or systemic sclerosis.

In one aspect, a method for treating ophthalmic disease (for example, Diabetic retinopathy (DR), Diabetic macular edema (DME), Choroidal neovascularization (CNV), Retinal vein occlusion (RVO), Central retinal vein occlusion (CRVO), Branch retinal vein occlusion (BRVO), or pathologic myopia (PM); preferably age-related macular degeneration (AMD)), cancer, renal fibrosis, cirrhosis, arthosclerosis, portal hypertension or systemic sclerosis is disclosed, the method comprising the administration of (an effective amount of) the protein as provided herein or as prepared by the method as disclosed herein above, the nucleic acid as provided herein, the vector as provided herein, the cell as provided herein, or the composition as provided herein, to a subject (in need of the treatment).

The cancer can be a solid cancer. The solid cancer can be colon cancer, hepatocellular carcinoma, non-small cell lung cancer, soft tissue sarcoma, prostate cancer, breast cancer, ovarian cancer, glioma, dermatofibrosarcoma protuberans, oral squamous cell carcinoma, or pancreatic cancer. The cancer can be a non-solid cancer, such as leukemia or non-Hodgkin's lymphoma.

The terms “treatment”, “treating” and the like are used herein to generally mean obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of partially or completely curing a disease and/or adverse effect attributed to the disease. The term “treatment” as used herein covers any treatment of a disease in a subject and includes: (a) preventing a disease related in a subject which may be predisposed to the disease; (b) inhibiting the disease, i.e. arresting its development; or (c) relieving the disease, i.e. causing regression of the disease.

An “individual”, “patient” or “subject” for the purposes of the present invention includes both humans and other animals, particularly mammals, and other organisms. Thus, the methods are applicable to both human therapy and veterinary applications. Preferably, the “individual”, “patient” or “subject” is a mammal, and most preferably the “individual”, “patient” or “subject” is human.

The protein provided herein may be administered as a single agent (i.e. in form of a monotherapy) or in form of a combination therapy, for example, conventional therapies of retinopathies like diabetic retinopathy, retinitis pigmentosa, dry/wet age related macular degeneration or glaucoma. Examples of cancers which may be treated by the present invention include those intra-axial brain cancers, ovarian cancers, colon cancers, prostate cancers, lung cancers, Kaposi's sarcoma and skin cancers, which have inappropriate PDGF-R activity. Examples of blood vessel proliferation disorders include, restenosis and atherosclerosis.

The pharmaceutical composition will be formulated and dosed in a fashion consistent with good medical practice, taking into account the clinical condition of the individual patient, the site of delivery of the pharmaceutical composition, the method of administration, the scheduling of administration, and other factors known to practitioners. The “effective amount” of the pharmaceutical composition for purposes herein is thus determined by such considerations.

The skilled person knows that the effective amount of pharmaceutical composition administered to an individual will, inter alia, depend on the nature of the compound. The administration of the herein provided compositions may, inter alia, comprise an administration twice daily, every day, every other day, every third day, every forth day, every fifth day, once a week, once every second week, once every third week, once every month, etc.

Pharmaceutical compositions of the invention preferably comprise a pharmaceutically acceptable carrier. By “pharmaceutically acceptable carrier” is meant a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The term “parenteral” as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

The pharmaceutical composition is also suitably administered by sustained release systems. Suitable examples of sustained-release compositions include semi-permeable polymer matrices in the form of shaped articles, e.g., films, or mirocapsules. Sustained-release matrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman, U. et al., Biopolymers 22:547-556 (1983)), poly (2-hydroxyethyl methacrylate) (R. Langer et al., J. Biomed. Mater. Res. 15:167-277 (1981), and R. Langer, Chem. Tech. 12:98-105 (1982)), ethylene vinyl acetate (R. Langer et al., Id.) or poly-D-(−)-3-hydroxybutyric acid (EP 133,988). Sustained release pharmaceutical composition also include liposomally entrapped compound. Liposomes containing the pharmaceutical composition are prepared by methods known per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. (USA) 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, the liposomes are of the small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol. percent cholesterol, the selected proportion being adjusted for the optimal therapy.

Generally, the formulations are prepared by contacting the components of the pharmaceutical composition uniformly and intimately with liquid carriers or finely divided solid carriers or both. Then, if necessary, the product is shaped into the desired formulation. Preferably the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution, and dextrose solution. Non aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes. The carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) (poly)peptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, manose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and/or nonionic surfactants such as polysorbates, poloxamers, or PEG.

The components of the pharmaceutical composition to be used for therapeutic administration must be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 micron membranes). Therapeutic components of the pharmaceutical composition generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

The components of the pharmaceutical composition ordinarily will be stored in unit or multi-dose containers, for example, sealed ampoules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution. As an example of a lyophilized formulation, 10-ml vials are filled with 5 ml of sterile-filtered 1% (w/v) aqueous solution, and the resulting mixture is lyophilized. The infusion solution is prepared by reconstituting the lyophilized compound(s) using bacteriostatic Water-for-Injection.

The nucleic acid molecule provided herein can also be employed alone or as part of a vector for gene therapy purposes. Gene therapy, which is based on introducing therapeutic genes into cells by ex vivo or in vivo techniques, is one of the most important applications of gene transfer. Suitable vectors, methods or gene delivery systems for in vivo gene therapy are described in the literature and are known to the person skilled in the art; see, e.g., Giordano (1996) Nat. Med. 2:534-539; Schaper (1996) Circ. Res. 79:911-919; Anderson (1992) Science 256:808-813; Verma (1997) Nature 389:239-249; Isner (1996) Lancet 348:370-374; Muhlhauser (1995) Circ. Res. 77:1077-1086; Onodera (1998) Blood 91:30-36; Verma (1998) Gene Ther. 5:692-699; Nabel (1997) Ann. N.Y. Acad. Sci. 811:289-292; Verzeletti (1998) Hum. Gene Ther. 9:2243-2251; Wang (1996) Nat. Med. 2:714-716; WO 94/29469; WO 97/00957, U.S. Pat. Nos. 5,580,859; 5,589,466; or Schaper (1996) Curr. Opin. Biotechnol. 7:635-640.

The nucleic acid molecules and vectors provided herein may be designed for direct introduction or for introduction via liposomes or viral vectors (e.g., adenoviral, retroviral) into the cell. For example, the vector can be an adeno-associated-virus (AAV) vector, in particular, an AAV8 vector. AAV vectors are attractive for gene therapy. The AAV system has several advantages including long-term gene expression, the inability to autonomously replicate without a helper virus, transduction of dividing and nondividing cells, and the lack of pathogenicity from wild-type infections. Preferably, said cell in which the nucleic acid molecule or vector is introduced is a germ line cell, embryonic cell or egg cell or derived therefrom, most preferably said cell is a stem cell. An example for an embryonic stem cell can be, inter alia, a stem cell as described in Nagy (1993) Proc. Natl. Acad. Sci. USA 90:8424-8428.

As used herein, the terms “comprising” or “including” or grammatical variants thereof are to be taken as specifying the stated features, integers, steps or components but do not preclude the addition of one or more additional features, integers, steps, components or groups thereof. The terms “comprising”/“including” encompass the terms “consisting of” and “consisting essentially of”. Thus, whenever the terms “comprising”/“including” are used herein, they can be replaced by “consisting essentially of” or, preferably, by “consisting of”.

The terms “comprising”/“including” mean that any further component (or likewise features, integers, steps and the like) can be present.

The term “consisting of” means that no further component (or likewise features, integers, steps and the like) can be present.

The term “consisting essentially of” or grammatical variants thereof when used herein are to be taken as specifying the stated features, integers, steps or components but do not preclude the addition of one or more additional features, integers, steps, components or groups thereof but only if the additional features, integers, steps, components or groups thereof do not materially alter the basic and novel characteristics of the claimed product, composition, device or method and the like.

Thus, the term “consisting essentially of” means that specific further components (or likewise features, integers, steps and the like) can be present, namely those not materially affecting the essential characteristics of the product, composition, device or method. In other words, the term “consisting essentially of” (which can be interchangeably used herein with the term “comprising substantially”), allows the presence of other components in the product, composition, device or method in addition to the mandatory components (or likewise features, integers, steps and the like), provided that the essential characteristics of the product, composition, device or method are not materially affected by the presence of other components.

The term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, biological and biophysical arts.

If not otherwise indicated, the term “about” as used herein refers to ±10%.

BRIEF DESCRIPTION OF THE FIGURES

The present invention is further described by reference to the following non-limiting figures and examples.

The Figures show:

FIG. 1 .

Nucleotide and amino acid sequence of the PDGFR_(αD123)-PAS(200)-VEGFR1_(D2)/R2_(D3) fusion protein referred to herein as EPS1108P encoded on pDSG33-PDGFR-PAS200-VEGFR (flanked by XbaI and HindIII restriction sites). Underlined: signal polypeptide sequence of PDGFR-α, which is cleaved off during secretion. Waved underlined: PAS polypeptide sequence. Broken underlined: His6 tag for affinity purification and detection.

FIG. 2 .

3D model of the fully ligand-bound complex of PDGFRα_(D123)-PAS(200)-VEGFR1_(D2)/R2_(D3) with both ligands, VEGF and PDGF, in its homo-dimeric state. For modelling the crystal structures of PDGFR-β in complex with PDGF-BB (PDB entry 3MJG) and VEGFR2 in complex with VEGF-C (PDB entry 2X1W) were used. The flexible PAS polypeptide spacer in a random coil conformation is depicted over-simplified as ribbon. (N or C=N- or C-terminal ending)

FIG. 3 .

Purification and SDS-PAGE analysis of the PDGFR_(αD12)3-PAS(200)-VEGFR1_(D2)/R2_(D3) fusion protein referred to herein as EPS1108P. (A) SDS-PAGE analysis of the different purification steps for PDGFRα_(D123)-PAS(200)-VEGFR1_(D)2/R2_(D3) transiently expressed in MEXi-293E cells after 7 days of transfection. (1) NH₄SO₄ precipitate from conditioned medium supernatant. (2) Protein after Resource Q (anion exchange) chromatography. (3) Protein after Resource S (cation exchange) chromatography. (4) Protein after size exclusion chromatography. Samples were analyzed on a 4-20% Gradient Bis-Tris Gel and visualized using InstantBlue colloidal Coomassie blue protein stain. Protein molecular weight marker: PageRuler Plus Prestained Protein Ladder (250, 130, 100, 70, 55, 35, 25, 15, 10 kDa). (B) SDS-PAGE analysis of PDGFRα_(D123)-PAS(200)-VEGFR1_(D2)/R2_(D3) purified from MEXi-293E conditioned medium under (1) reduced and (2) unreduced conditions (+/−5 mM DTT). (C) Western blot analysis of purified PDGFR_(αD123)-PAS(200)-VEGFR1_(D2)/R2_(D3) via the C-terminal His6-tag using an anti-polyHis antibody. Protein molecular weight marker: PageRuler Plus Prestained Protein Ladder (250, 130, 100, 70, 55, 35, 25, 15, 10 kDa).

FIG. 4 .

Size exclusion chromatography (SEC) analysis of PDGFR_(αD123)-PAS (200)-VEGFR1_(D2)/R2_(D3) referred to herein as EPS1108P on Superdex 200 10/30 GL (running buffer: 10 mM Hepes/NaOH, 150 mM NaCl pH 7.4; void volume V₀=7.1 ml; column volume: 23.6 ml; sample volume: 0.5 ml). (A) PDGFR_(αD123)-PAS(200)-VEGFR1_(D2)/R2_(D3) purified from conditioned MEXi-293E medium elutes at 9.6 ml as a sharp peak. (B) The calibration line used to estimate the native molecular weight based on the retention volumes of various globular size standard proteins during analytical gel filtration on the same Superdex 200 10/30 GL column. Calculated from the semi-logarithmic fit, the PASylated fusion protein reveals an apparent molecular weight of approximately 530 kDa, which is 7-fold larger than the calculated molecular mass based on amino acid sequence (75 kDa) of PDGFR_(αD123)-PAS(200)-VEGFR1_(D2)/R2_(D3), thus illustrating the expanded molecular volume due to the random coil behavior of the PAS spacer.

FIG. 5 .

Electromobility gel shift assay via native PAGE of PDGFR_(αD123)-PAS(200)-VEGFR1_(D2)/R2_(D3) referred to herein as EPS1108P in the presence of equimolar amounts of either PDGF-AA or VEGF-A165 or both. (1) The PDGFR_(αD123)-PAS(200)-VEGFR1_(D2)/R2_(D3) fusion protein purified from conditioned MEXi-293E medium is glycosylated and runs as a broad band according to a calculated mass of 72.3 kDa based on the amino acid sequence (without glycosylation). Binding of 38.4 kDa homodimeric VEGF-A165 (3), 28.6 kDa homodimeric PDGF-AA (4) or both protein ligands, VEGF-A165 and PDGF-AA (2), considerably changes the electrophoretic migration behavior of PDGFR_(αD123)-PAS(200)-VEGFR1_(D2)/R2_(D3) and also leads to a more focused and defined protein band, which is indicative of the complexes formed.

FIG. 6 .

SDS-PAGE analysis result of purified PDGFRα_(D123)-PAS (300)-VEGFR1_(D2)/R2_(D3), referred to herein as EPS1103P, was shown in FIG. 6A. SEC analysis of purified EPS1103P protein was shown in FIG. 6B, which showed a purity of 98.88%.

FIG. 7 .

SDS-PAGE analysis result of purified PDGFRα_(D123)-PAS(400)-VEGFR1_(D2)/R2_(D3), referred to herein as EPS1104P, was shown in FIG. 7A. SEC analysis of purified EPS1104P protein was shown in FIG. 7B, which showed a purity of 98.97%.

FIG. 8 .

SDS-PAGE analysis result of purified VEGFR1_(D2)/R2_(D3)-PAS(200)-PDGFRα_(D123), referred to herein as EPS1105P, was shown in FIG. 8A. SEC analysis result of purified EPS1105P protein was done, the result was shown in FIG. 8B, which showed a purity of 99.82%.

FIG. 9 .

SDS-PAGE analysis result of purified PDGFRα_(D123)-(GGGGS (SEQ ID NO: 73))₃-PAS(200)-(GGGGS (SEQ ID NO: 73))₃-VEGFR1D2/R2D3 referred to herein as EPS1106P, was shown in FIG. 9A. SEC analysis of purified EPS1106P protein was done, the result was shown in FIG. 9B, which showed a purity of 99.79%.

FIG. 10 .

SDS-PAGE analysis result of purified VEGFR1_(D2)/R2_(D3)-(GGGGS (SEQ ID NO: 73))₃-PAS(200)-(GGGGS (SEQ ID NO: 73))₃-PDGFRαD123 referred to herein as EPS1107P, was shown in FIG. 10A SEC analysis of purified EPS1107P protein was done, the result was shown in FIG. 10B, which showed a purity of 99.43%.

FIG. 11 .

SDS-PAGE analysis result of purified PAS(200)-VEGFR1_(D2)/R2_(D3)-PDGFRα_(D123) referred to herein as EPS1109P, was shown in FIG. 11A. SEC analysis of purified EPS1109P protein was done, the result was shown in FIG. 11B, which showed a purity of 99.62%.

FIG. 12 .

SDS-PAGE analysis result of purified PAS(200)-PDGFRα_(D123)-VEGFR1_(D2)/R2_(D3), referred to herein as EPS1110P, was shown in FIG. 12A. SEC analysis of purified EPS1110P protein was done, the result was shown in FIG. 12B, which showed a purity of 99.52%.

FIG. 13 .

SDS-PAGE analysis result of purified PDGFRαD₁₂₃-PAS(600)-VEGFR1_(D2)/R2_(D3), referred to herein as EPS1113P, was shown in FIG. 13A. SEC analysis of purified EPS1113P protein was done, the result was shown in FIG. 13B, which showed a purity of 92.28%.

FIG. 14 .

SDS-PAGE analysis result of purified PDGFRα_(D123)-(GGGGS)₃-PAS(600)-(GGGGS)₃-VEGFR1_(D2)/R2_(D3), referred to herein as EPS1114P, was shown in FIG. 14A. SEC analysis of purified EPS1114P protein was done, the result was shown in FIG. 14B, which showed a purity of 98.77%.

FIG. 15 .

SDS-PAGE analysis result of purified PDGFRαD123-(GGGGS (SEQ ID NO: 73))₃-PAS(600)-(GGGGS (SEQ ID NO: 73))₃-VEGFR1_(D2)/R2_(D3), referred to herein as EPS1114P, was shown in FIG. 15A. SEC analysis of purified EPS1114P protein was done, the result was shown in FIG. 15B, which showed a purity of 98.77%.

FIG. 16 .

SDS-PAGE analysis result of purified VEGFR1_(D2)/R2_(D3)-(GGGGS (SEQ ID NO: 73))₃-PAS(600)-(GGGGS (SEQ ID NO: 73))₃-PDGFRα_(D123), referred to herein as EPS1115P, was shown in FIG. 16A. SEC analysis of purified EPS1115P protein was done, the result was shown in FIG. 16B, which showed a purity of 99.58%.

The Examples illustrate the invention.

Example 1: Cloning of PDGFRα_(D123)-PAS(200)-VEGFR1_(D2)/R2_(D3)

PDGFRα_(D123)-PAS(200)-VEGFR1_(D2)/R2_(D3) is referred to herein as EPS1108P.

The DNA sequence encoding the fusion protein PDGFRα_(α123)-PAS(200)-VEGFR1_(D2)/R2_(D3) was constructed in two steps. First, the coding region for the two receptor ectodomains was obtained by gene synthesis from Geneart (Regensburg, Germany; SEQ ID No. 17). In this construct, (i) the DNA sequence coding for the PDGFR-α leader signal polypeptide sequence (69 bp, including the start Met) is followed by (ii) the 876 bp nucleotide sequence for the PDGF-receptor a domains D1-D3, (iii) the 615 bp sequence for VEGFR1_(D2)/VEGFR2_(D3), (iv) a His₆-tag and, finally, a stop-codon. Restriction sites for SapI were introduced between the coding regions for PDGFR-α_(D123) and VEGFR1_(D2)/VEGFR2_(D3) to allow subsequent in-frame cloning of a PAS or P/A sequence serving as flexible linker/spacer. In addition, restriction sites for XbaI and HindIII were introduced at the flanking regions of the entire synthetic gene to simplify cloning onto expression vectors with compatible restriction endonuclease sites. Note that a naturally occurring restriction site for XbaI within the gene of PDGFR-α is sensitive to dam methylation and is blocked for restriction digest with XbaI when using plasmid DNA prepared from a dam⁺ host bacterium such as E. coli strain XL1Blue. Nucleotide sequences for the receptor ectodomains were taken from Genbank entry NM006206.4 for PDGFR-α_(D123) and from U.S. Pat. No. 5,952,199 for VEGFR1_(D2)/R2_(D3) (Aflibercept). The full length synthetic gene (990 bp) was cloned via the XbaI/HindIII sites on pDSG33, a derivative of pDSG-IBA33 (IBA, Göttingen, Germany), designed for high-level stable and non-replicative transient expression in mammalian host cells. In the second step, a gene fragment encoding a PAS sequence of 200 residues was excised from the plasmid pXL1-PAS(200) via double cut with the restriction enzyme SapI and inserted into the pDSG33 vector with the cloned synthetic gene, which had been linearized with SapI. After analytical restriction digest and confirmation of the correct insert via DNA sequencing (MWG, Ebersberg, Germany) the resulting expression plasmid encoding the PDGFRα_(D123)-PAS(200)-VEGFR1_(D2)/R2_(D3) fusion protein (SEQ ID No. 18; SEQ ID No. 14; FIG. 1 ) was designated as pDSG33-PDGFR-PAS200-VEGFR (SEQ ID No. 13).

Example 2: Expression of PDGFRα_(D123)-PAS(200)-VEGFR1_(D2)/R2_(D3)

For production of milligram quantities of the fusion protein (FIG. 1 ) plasmid DNA of pDSG33-PDGFR-PAS200-VEGFR (SEQ ID No. 13) was prepared using the QIAGEN Plasmid Midi Kit (Qiagen, Hilden, Germany) and then used to transfect 200 ml of exponentially growing MEXi-293E suspension cells (IBA, Gottingen, Germany) in MEXi-TM Transfection medium (IBA; supplemented with 8 mM L-Alanyl-L-Glutamin). Transfection was accomplished according to the manufacturer's instructions using polyethylenimine (PEI; Polyscienences, Warrington Pa., USA) and plasmid DNA at a mass ratio of 4 to 1 and applying 1 μg DNA per one million cells at a density of 1×10⁶ cells/ml. Four hours after transfection, cells were diluted in fresh MEXi-CM cultivation medium (IBA; supplemented with 50 mg/l G-418 and 8 mM L-Alanyl-L-Glutamin) to a final culture volume of 400 ml. The transfected cells were incubated for 7 days under mild agitation, at 120 rpm, at 37° C. in a humified CO₂—Incubator. After that, cells were removed by centrifugation at 4500 g for 20 min and the cleared conditioned medium containing the PDGFRα_(D123)-PAS(200)-VEGFR1_(D2)/R2_(D3) fusion protein, referred to herein as EPS1108P was collected and sterile filtered (0.2 μm).

Example 3: Protein Purification of PDGFRα_(D123)-PAS(200)-VEGFR1_(D2)/R2_(D3)

PDGFRα_(D123)-PAS(200)-VEGFR1_(D2)/R2_(D3) (FIG. 1 ), referred to herein as EPS1108P was precipitated from the cleared culture medium obtained above by adding 150 g of ammonium sulphate to 400 ml of the conditioned medium. The mixture was incubated over night at 4° C. under gentle stirring, then the precipitate was collected by centrifugation at 15.000 g for 40 min. The pellet was recovered and dissolved in 100 ml 40 mM Hepes/NaOH, pH 7.4 containing 1 M NaCl and dialyzed against the same buffer over night at 4° C. For immobilized metal ion affinity chromatography (IMAC), a 6 ml HisTrap HP column (GE Healthcare, Uppsala, Sweden) was equilibrated with 100 ml 40 mM Hepes/NaOH, pH 7.4, 1 M NaCl (running buffer) and approximately 100 ml of the sterile-filtered protein solution was loaded. The column was washed with the same buffer until absorbance at 280 nm (A₂₈₀) reached baseline and PDGFRα_(D123)-PAS(200)-VEGFR1_(D2)/R2_(D3) was eluted using a linear gradient of 0 to 210 mM imidazole/HCl in running buffer over 8 column volumes. For subsequent anion exchange chromatography, the elution fraction containing PDGFRα_(D123)-PA S(200)-VEGFR1_(D2)/R2_(D3) was dialyzed against chromatography buffer (20 mM MES/NaOH, pH 5.9) over night at 4°, sterile-filtered and then loaded on a pre-equilibrated Resource Q column (GE Healthcare, Uppsala, Sweden) with a bed volume of 85 ml. The column was washed with chromatography buffer to A₂₈₀ baseline before the fusion protein was eluted in one step by buffer change to chromatography buffer supplemented with 225 mM NaCl. In the eluted fraction the fusion protein was about 85% pure. As a final polishing step, this protein solution was dialysed against 20 mM MES/NaOH, pH 5.9 overnight and loaded on a Resource S column (GE Healthcare) with 85 ml bed volume and equilibrated with the same buffer. Elution was achieved by applying a step-wise concentration gradient, in the same buffer, starting with 150 mM NaCl followed by 225 mM NaCl, and 300 NaCl, finally yielding the fusion protein. The purity of the PDGFRα_(D123)-PAS(200)-VEGFR1_(D2)/R2_(D3) was analysed by SDS-PAGE (FIG. 3 ) using 4-20% Bis-Tris gradient gels (Genscript, Piscataway N.J., USA) in MOPS running buffer according to the manufacturer's instructions, followed by staining with InstantBlue colloidal Coomassie blue protein stain (Expedeon, Cambridge, UK). The gels were documented by digital imaging. Note: the apparently higher molecular weight of the decoy receptor fusion seen in SDS-PAGE (FIG. 3 ) results from PASylation, which has already been observed for other PASylated proteins in Schlapschy et al., 2013.

Example 4: Western Blot Analysis of PDGFRα_(D123)-PAS(200)-VEGFR1_(D2)/R2_(D3)

Purified PDGFRα_(D123)-PAS(200)-VEGFR1_(D2)/R2_(D3), referred to herein as EPS1108P, carrying a C-terminal His₆-tag, was separated on a 4-20% SDS Bis-Tris gradient gel (Genscript) in MOPS running buffer according to the manufacturer's instructions and blotted onto an Immobilion-P PVDF membrane (Merck, Darmstadt, Germany) using a semi-dry transfer apparatus. The membrane was washed twice with phosphate-buffered saline (PBS; 4 mM KH₂PO₄, 16 mM Na₂HPO₄, 115 mM NaCl pH 7.4) supplemented with 0.1% Tween-20 (PBST) and then blocked for unspecific binding with a solution of 3% (w/v) BSA in PBST for 1 h at room temperature (RT). Next, the blocked membrane was incubated in a solution of Monoclonal Anti-polyHistidine-Peroxidase clone HIS-1 antibody conjugate (A7058; Sigma Aldrich, St. Louis, Mo., USA), diluted to 1:2000 in 0.1% (w/v) BSA, PBST for 1 h at RT. The membrane was washed twice with PBST and then the horseradish peroxidase substrate 3,3′-diaminobenzidine (Sigma Aldrich) was added. At the size of PDGFRαD₁₂₃-PAS(200)-VEGFR1_(D2)/R2_(D3) a brownish precipitate was detected on the membrane, which was documented by digital imaging (FIG. 3 ).

Example 5: Size Exclusion Chromatography of PDGFRα_(D123)-PAS(200)-VEGFR1_(D2)/R2_(D3)

For analyzing the integrity and apparent size of the purified PDGFRα_(D123)-PAS(200)-VEGFR1_(D2)/R2_(D3), referred to herein as EPS1108P, 500 μl of a 0.43 mg/ml protein sample (3 nmol) in 20 mM MES/NaOH, pH 5.9, 300 mM NaCl was loaded on a Superdex 200 10/30 GL column (GE Healthcare) that was pre-equilibrated with 10 mM Hepes/NaOH, pH 7.4, 150 mM NaCl. Purified PDGFRα_(D123)-PAS(200)-VEGFR1_(D2)/R2_(D3) from conditioned MEXi-293E medium as described above elutes at 9.6 ml as a sharp peak (FIG. 4 , A), which corresponds to an average molecular weight of 530 kDa, as calculated from the calibration curve (FIG. 4 , B).

Example 6: Native PAGE and Electromobility Gel Shift Assay

Purified PDGFRα_(D123)-PAS(200)-VEGFR1_(D2)/R2_(D3), referred to herein as EPS1108P (25 pmol) was incubated with either 25 pmol VEGF-A₁₆₅ (#8065-LF; Cell Signaling Technology, Danvers Mass., USA) or 25 pmol PDGF-AA (#8913-LF; Cell Signaling Technology) or both ligands (each 25 pmol), as indicated in FIG. 5 , in 25 μl reactions in the presence of 20 mM HEPES/NaOH, pH 7.4, 100 mM NaCl for 30 min on ice. The solutions were then mixed with 10× native sample buffer (60 mM Tris base, 480 mM glycine, pH 8.3; 50% (v/v) glycerol, 0.01% (w/v) bromophenol blue) and immediately loaded onto a 3-8% Tris-acetate polyacrylamide gel (without SDS; Invitrogen, Carlsbad, Calif., USA). The gel was run at 90 V in Tris-glycine native running buffer, pH 8.3 (Invitrogen) at RT until the bromophenol blue marker reached the bottom of the gel. The gel was shortly rinsed in water and then stained using InstantBlue colloidal Coomassie blue protein stain (Expedeon, Cambridge, UK). The gel was documented by digital imaging. Under native conditions used for PAGE, both ligands, VEGF-A165 and PDGF-AA, bind to PDGFRαD123-PAS(200)-VEGFR1D2/R2D3 and result in stable complexes (ref. FIG. 2 ), that can be detected: (I) Simultaneous binding of both ligands, when both ligands are present or (II) binding of either PDGF-AA or VEGF-A165, in the absence of the other ligand (FIG. 5 ).

Example 7 Cloning, Expression and Purification of PDGFRα_(D123)-PAS (300)-VEGFR1_(D2)/R2_(D3)

PDGFRα_(pin)-PAS (300)-VEGFR1_(D2)/R2_(D3) is referred to herein as EPS1103P.

Cloning of EPS1103P:

PCR primer and the sequencing primer of EPS1103P were designed, and the gene was synthesized de novo. The gene was amplified based on the PCR primer and then ligated into vector pUC57. The vectors were transfected into E. coli competent cells, which were incubated at 37° C. overnight; the positive clones were identified by colony PCR screening; and the plasmids from the positive clones were extracted for verification of the correct insert. The extracted plasmids and the target vector (pcDNA3.4) were digested by restriction enzyme; the digested products were recovered by electrophoresis, and then ligated by ligase in buffer; the buffer solution was incubated at 37° C. overnight; the positive clones were identified by colony PCR screening; and the plasmids from the positive clones were extracted for verification of the correct insert.

An aliquot of the above plasmids were digested by enzyme in a thermostatic water bath, and then the mixture was verified by agarose gel electrophoresis; the verified plasmids were transfected into E. coli; an aliquot of the bacteria culture were spread on solid LB plate with resistance for screening; then the bacteria clones were amplified in medium overnight in an incubator; the plasmids were extracted from the positive clones.

Expression of EPS1103P:

CHO-3E7 cells were grown in serum-free FreeStyle™ CHO Expression Medium (Life Technologies, Carlsbad, Calif., USA). The cells were maintained in Erlenmeyer Flasks (Corning Inc., Acton, Mass.) at 37° C. with 5% CO₂ on an orbital shaker (VWR Scientific, Chester, P A). Two days before the transfection, the cells were seeded at an appropriate density. On the day of transfection, the plasmid and transfection reagent were mixed at an optimal ratio and then added into the flask with cells. The cell culture supernatants collected on day 6 were used for purification.

Purification of EPS1103P:

The above cell culture broth was centrifuged and followed by filtration. The filtered supernatants were diluted with 25 mM Tris-HCl buffer, pH8.0, then loaded onto a Hitrap Q HPColumn (GE, Cat. No. 17115401) at 1.0 ml/min, after washing and elution with appropriate buffer, the eluted fractions were pooled and purified by Ni column (GenScript, Cat. No. L00465). The target protein was further purified via HiLoad Superdex 200 26/600 pg column (GE Healthcare, Uppsala, Sweden) to remove HMW aggregation and other impurities. The purified proteins were analyzed by SDS-PAGE and SEC-HPLC by using standard protocols for molecular weight and purity measurements as shown in FIG. 6A and FIG. 6B; the result of SEC-HPLC showed a purity of 98.88%.

Example 8: Cloning, Expression and Purification of PDGFRα_(D123)-PAS (400)-VEGFR1_(D2)/R2 D₃

PDGFRα_(D123)-PAS(400)-VEGFR1_(D2)/R2_(D3) is referred to herein as EPS1104P. The Cloning, Expression and Purification procedures of EPS1104P were referred to Example 7. SDS-PAGE analysis result of purified EPS1104P protein was shown in FIG. 7A, and the SEC analysis of purified EPS1104P protein was done, the result was shown in FIG. 7B, which showed a purity of 98.97%.

Example 9: Cloning, Expression and Purification of VEGFR1_(D2)/R2_(D3)-PAS(200)-PDGFRα_(D123)

VEGFR1_(D2)/R2_(D3)-PAS(200)-PDGFRα_(D123) is referred to herein as EPS1105P. The Cloning, Expression and Purification procedures of EPS1105P were referred to Example 7. SDS-PAGE analysis result of purified EPS1105P protein was shown in FIG. 8A, and SEC analysis of purified EPS1105P protein was done, the result was shown in FIG. 8B, which showed a purity of 99.82%.

Example 10: Cloning, Expression and Purification of PDGFRα_(D123)-(GGGGS)₃-PAS(200)-(GGGGS)₃-VEGFR1_(D2)/R2_(D3)

PDGFRα_(D123)-(GGGGS)₃-PAS(200)-(GGGGS)₃-VEGFR1_(D2)/R2_(D3) is referred to herein as EPS1106P. The Cloning, Expression and Purification procedures of EPS1106P were referred to Example 7. SDS-PAGE result analysis of purified EPS1106P protein was shown in FIG. 9A, and SEC analysis of purified EPS1106P protein was done, the result was shown in FIG. 9B, which showed a purity of 99.79%.

Example 11: Cloning, Expression and Purification of VEGFR1_(D2)/R2_(D3)-(GGGGS)₃-PAS(200)-(GGGGS)₃-PDGFRα_(D123)

VEGFR1_(D2)/R2_(D3)-(GGGGS)₃-PAS(200)-(GGGGS)₃-PDGFRα_(D123) is referred to herein as EPS1107P. The Cloning, Expression and Purification procedures of EPS1107P were referred to Example 7. SDS-PAGE analysis result of purified EPS1107P protein was shown in FIG. 10A, and SEC analysis of purified EPS1107P protein was done, the result was shown in FIG. 10B, which showed a purity of 99.43%.

Example 12: Cloning, Expression and Purification of PAS(200)-VEGFR1_(D2)/R2_(D3)-PDGFRα_(D123)

PAS(200)-VEGFR1_(D2)/R2_(D3)-PDGFRα_(D123) is referred to herein as EPS1109P. The Cloning, Expression and Purification procedures of EPS1109P were referred to Example 7, respectively. SDS-PAGE analysis of result purified EPS1109P protein was shown in FIG. 11A, and SEC analysis of purified EPS1109P protein was done, the result was shown in FIG. 11B, which showed a purity of 99.62%.

Example 13: Cloning, Expression and Purification of PAS(200)-PDGFRα_(D123)-VEGFR1_(D2)/R2_(D3)

PAS(200)-PDGFRα_(D123)-VEGFR1_(D2)/R2_(D3) is referred to herein as EPS1110P. The Cloning, Expression and Purification procedures of EPS1110P were referred to Example 7. SDS-PAGE analysis result of purified EPS1110P protein was shown in FIG. 12A, and SEC analysis of purified EPS1110P protein was done, the result was shown in FIG. 12B, which showed a purity of 99.52%.

Example 14: Cloning, Expression and Purification of PDGFRβ_(D123)-PAS(200)-VEGFR1_(D2)/R2_(D3)

PDGFRβ_(D123)-PAS(200)-VEGFR1_(D2)/R2_(D3), is named herein as EPS1111P. Its Cloning, Expression and Purification procedures refer to the described method of Example 7.

Example 15: Cloning, Expression and Purification of PDGFRαD₁₂₃-PAS(600)-VEGFR1_(D2)/R2_(D3)

PDGFRαD₁₂₃-PAS(600)-VEGFR1_(D2)/R2_(D3) is referred to herein as EPS1113P. The Cloning, Expression and Purification procedures of EPS1113P were referred to Example 7. SDS-PAGE analysis result of purified EPS1113P protein was shown in FIG. 13A, and SEC analysis of purified EPS1113P protein was done, the result was shown in FIG. 13B, which showed a purity of 92.28%.

Example 16: Cloning, Expression and Purification of PDGFRα_(D123)-(GGGGS)₃-PAS(600)-(GGGGS)₃-VEGFR1_(D2)/R2_(D3)

PDGFRα_(D123)-(GGGGS)₃-PAS(600)-(GGGGS)₃-VEGFR1_(D2)/R2_(D3) is referred to herein as EPS1114P. The Cloning, Expression and Purification procedures of EPS1114P were referred to Example 7. SDS-PAGE analysis result of purified EPS1114P protein was shown in FIG. 14A, and SEC analysis of purified EPS1114P protein was done the result was shown in FIG. 14B, which showed a purity of 98.77%.

Example 17: Cloning, Expression and Purification of VEGFR1_(D2)/R2_(D3)-(GGGGS)₃-PAS(600)-(GGGGS)₃-PDGFRα_(D123)

VEGFR1_(D2)/R2_(D3)-(GGGGS)₃-PAS(600)-(GGGGS)₃-PDGFRα_(D123) is referred to herein as EPS1115P. The Cloning, Expression and Purification procedures of EPS1115P were referred to Example 7. SDS-PAGE analysis result of purified EPS1115P protein was shown in FIG. 15A, and SEC analysis of purified EPS1115P protein was done, the result was shown in FIG. 15B, which showed a purity of 99.58%.

Example 18: Cloning, Expression and Purification of Mutant PDGFRα_(D123)-PAS(200)-VEGFR1_(D2)/R2_(D3)

Mutant PDGFRα_(D123)-PAS(200)-VEGFR1_(D2)/R2_(D3), is named herein as EPS1116P. Its Cloning, Expression and Purification procedures refers to the described method of Example 7.

Example 19: Binding Affinity with VEGF₁₆₅/PDGF-BB Ligands

1. Assay Method

To detect the affinity with VEGF, the test fusion proteins and the reference were serially diluted with reagent dilution solution respectively, mixed with human VEGF₁₆₅ ligand (final concentration was 50 μM), and incubated for 1 hour at room temperature on a shaker set at 300 RPM. The amount of unbound VEGF₁₆₅ was then measured by a human VEGF-specific ELISA (Human VEGF DuoSet ELISA kit, R&D Systems, CAT. No. DY293B-05).

To detect the affinity with PDGF-BB, the test fusion proteins and the reference were serially diluted with reagent dilution solution respectively, mixed with human PDGF-BB ligand (final concentration was 1 ng/ml), and incubated for 1 hour at room temperature on a shaker set at 300 RPM. The amount of unbound PDGF-BB was then measured by a human PDGF-BB-specific ELISA (Human PDGF-BB DuoSet ELISA kit, R&D Systems, CAT. No. DY220).

2. Assay Procedure

2.1 Reagents Preparation

2.1.1 Coating Buffer

PBS: 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na₂HPO₄, 1.5 mM KH₂PO₄, pH 7.2-7.4, filtered through a 0.2 μm filter.

2.1.2 Washing Buffer

Dissolved 9.55 g PBS power into Milli-Q water, and brought to the total volume up to 1 L that contained 0.05% Tween 20 (v/v), and adjusted pH to 7.4.

2.1.3 Blocking Buffer

3 g of Bovine Serum Albumin (BSA) was added into 100 mL of PBS.

2.1.4 Reagent Dilution Solution

1 g of Bovine Serum Albumin (BSA) was added into 100 mL of PBS.

2.1.5 Stop Solution

81.4 mL of 36-38% hydrochloric acid was added to Mill-Q water, and brought the total volume up to 1 L.

2.2. Procedure

2.2.1 Plate Coating

Diluted the captured antibody using PBS to the working concentration (400 ng/mL), which was transferred into a 96-well microplate with 100 μL per well immediately. Sealed the plate and incubated at room temperature overnight.

2.2.2 Washing

Aspirated each well and washed with washing buffer (300 μL), repeating this process twice.

2.2.3 Blocking

Blocked the plates by adding 300 μL blocking buffer to each well, incubated at room temperature for 1 hour.

2.2.4 Sample Preparation and Pre-Incubation

To prepare affinity samples, the test fusion proteins (EPS1103P, EPS1104P, EPS1105P, EPS1106P, EPS1107P, EPS1108P, EPS1109P, EPS1110P, EPS1111P, EPS1113P, EPS1114P, EPS1115P or EPS1116P) or Reference (Affibercept) were serially diluted with reagent dilution solution respectively, mixed with human VEGF₁₆₅ ligand (final concentration was 50 pM) or human PDGFBB ligand (final concentration was 1 ng/ml), incubated for 1 hour at room temperature on a shaker set at 300 RPM.

To prepare the standard samples, PDGF-BB or VEGF₁₆₅ was diluted using 2-fold serial dilutions with reagent dilution solution (2000, 1000, 500, 250, 125, 62.5 and 31.25 pg/ml), respectively.

2.2.5 Sample Incubation

100 μL of the sample solution per well was transferred into the coated assay plate, all samples were duplicated. The assay plates were covered with acetate plate sealers, and the plates were incubated for 2 hour at room temperature on the shaker set at 500 rpm, washing the plate three times.

2.2.6 Incubation with Detection Antibody

100 μL of the diluted detection antibody was added into each well of the plate, which was then covered with a new adhesive strip and incubated for 1 hour at RT on the shaker set at 500 rpm; washing the plate three times.

2.2.7 Incubation with Streptavidin-HRP

100 μL of the pre-prepared Streptavidin-HRP solution was added into each well, which was then covered with a new adhesive strip and incubated for 30 minutes at room temperature; washing the plates three times.

2.2.8 Incubation with Substrate Solution (TMB)

100 μL of substrate solution was added into each well, incubated for 10 minutes at room temperature.

2.2.9 Adding Stop Solution (1N HCl)

After incubation with TMB for 10 minutes, 100 μL of stop solution (1 N HCL) was added to each well, the plate was gently tapped to ensure thoroughly mix.

2.2.10 Plate Reading

The optical density of each well was determined immediately, using Molecular Devices M2E plate reader with SoftMax Pro 6.5.1 GxP set at 450 nm and 570 nm; readings at 570 nm were subtracted from the readings at 450 nm to give the optical density of each well.

2.2.11 Data Analysis

Unbound human VEGF₁₆₅ or PDGF-BB was calculated using 4 parameters curve with the absorbance value. And the IC₅₀ of the tested fusion proteins and the reference was calculated using 4 parameters curve with unbound human VEGF₁₆₅ or PDGF-BB.

3. Result

TAB 1 Binding affinity with VEGF₁₆₅/PDGFBB ligands (IC₅₀) Human Human Analyte VEGF₁₆₅ (M) PDGFBB (M) Aflibercept 9.82E−12 —* EPS1108P 3.20E−10 6.63E−8 EPS1103P 8.69E−10 TBD EPS1104P 4.95E−10 TBD EPS1105P 5.46E−10 TBD EPS1106P 5.55E−10 TBD EPS1107P 3.04E−10 TBD EPS1109P 2.18E−10 TBD EPS1110P 3.31E−10 TBD EPS1111P TBD TBD EPS1113P 6.46E−10 TBD EPS1114P 5.06E−10 TBD EPS1115P 4.40E−10 TBD EPS1116P TBD TBD *No binding affinity was detected. 4. Conclusion

High binding affinity with human VEGF₁₆₅ ligand was observed for both the tested fusion proteins and the reference; while only the fusion proteins could bind with human PDGFBB, and the affinity is strong.

Example 20: Inhibition of VEGF-induced HUVEC Proliferation

1. Assay Methods

1.1 Three groups were designed, they are including Blank control, Model control (VEGF control) and Test articles (TAs) groups. Samples were tested in triplicate, and repeated the tests three times.

1.2 HUVEC cells growing in an exponential growth phase were harvested and prepared for single cell suspension.

1.3 The cells were counted and adjusted to the concentration of 5×10⁴ cells/mL with basal Medium. 100 μL of the cell suspension was seeded into the 96 well plate, or 100 μL of PBS was added (Blank control). Incubated at 37° C., 5% CO₂ overnight (without feed). 1.4 TAs were serially diluted to working concentration with assay medium (mixed by Complete Medium and Basal Medium) containing VEGF₁₆₅, working concentration was determined by pre-experiment, and the final concentration of VEGF₁₆₅ was 25 ng/ml. 100 μL of diluted TAs were added into the 96 well plate, incubated for 72 h at 37° C., 5% CO₂. 1.5 After incubation, 20 μL of Cell Counting Kit-8 was added into each well of the 96 well plate, then incubated in incubator for 4-6 h. 1.6 Absorbance (OD value) was measured at 450 nm with a microplate reader. 1.7 The IC₅₀ in each group was calculated with graphpad prism 5 software (GraphPad Software, Inc). 2. Results

TAB 2 Inhibition of HUVEC cells proliferation in each group Samples IC₅₀(nM) EPS1108P 28.49 EPS1105P 28.45 EPS1106P 39.67 EPS1107P 53.11 3. Conclusion

High inhibition potency to VEGF₁₆₅-induced HUVEC cell proliferation were observed for all TAs (EPS1108P, EPS1105P, EPS1106P and EPS1107P).

Example 21: Inhibition of Intersegmental Vessels (ISVs) Development in Zebrafish

1. Methods

Angiogenesis leads to formation of the intersegmental vessels (ISVs) of zebrafish embryo trunk, thus it has been utilitized as a human disease model to investigate the effect of testing compounds.

Procedure:

Collected Tg(Flka-GFP) transgenic zebrafish embryos at 28 hpf and removed the chorion with Proteinase E of Streptomyces griseus (Biology Institute of Shandong Academy of Sciences). Chose normal embryos under a stereomicroscope, anesthetized the embryos in a fresh prepared fish water containing 200 μg/mL tricaine, and then 10 nanoliters of the tested fusion proteins (500, 250, 25 or 2.5 μg/ml, respectively) were injected into the yolk sac of the zebrafish embryos by using an electronically regulated air-pressure microinjector; then the zebrafish embryos were transferred into a 24 well plate, 8-10 embryos per well; three paralleled wells for each testing sample group. The plate were covered and incubated at 28° C. in an illuminating incubator. Anesthetized embryos were observed and photographed under a fluorescence stereomicroscope at 48 hpf, vessel length of ISVs was measured, meanwhile observing the mortality and abnormality of the embryos.

2. Result

No mortality and abnormality were observed in any group, and the everage vessel length of ISVs in each group was listed in the table below (table 3):

TABLE 3 Inhibition of intersegmental vessels (ISVs) development in zebrafish vessel length Fusion Concentration of ISVs protein (μg/mL) (μm) Vehicle Control — 3299.05 ± 204.81  EPS1108P 250 2314.25 ± 85.37**  EPS1104P 250 2493.25 ± 141.37** EPS1107P 250 2514.25 ± 125.47** EPS1113P 250 2719.59 ± 238.38*  500 2446.46 ± 368.47** EPS1114P 250 2696.03 ± 179.86*  500 2426.69 ± 324.37** EPS1115P 500 2511.79 ± 418.55** *Compared with Vehicle Control group, the difference is significant (p < 0.05); **Compared with Vehicle Control group, the difference is significant (p < 0.01). 3. Conclusion

Compared with the Tg(Flk1-GFP) transgenic zebrafish embryos of vehicle control group, a significant decrease of vessel length was observed (p<0.01) in EPS1104P, EPS1107P, EPS1108P, EPS1113P, EPS1114P and EPS1115P groups, the results indicated that the tested fusion proteins (EPS1104P, EPS1107P, EPS1108P, EPS1113P, EPS1114P and EPS1115P) can significantly inhibit the intersegmental vessels (ISVs) development in zebrafish embryos.

Example 22: Inhibition of Tumor Neovascularization in Zebrafish

1. Methods

In the present study, a novel xenograft tumor model in Tg(Flk1:EGFP) transgenic zebrafish was established, in which individual green endothelial cells can be clearly distinguished from red tumor cells. This model can be used to investigate the inhibition effect of antiangiogenic compounds on tumor neovascularization.

Procedure:

1.1 Establishment of Xenograft Tumor Model in Zebrafish

B16-F10-mCherry tumor cells were transfected with pcDNA3.1 plasmids or pcDNA3.1 plasmids encoding human VEGFA, the cells were cultured and harvested at 48 h, and 10 nanoliters suspension containing about 200 cells were implanted into each Tg(Flk1-GFP) transgenic zebrafish embryo (State Key Laboratory of Biotherapy, Sichuan University, China) through the perivitelline space by using an electronically regulated air-pressure microinjector.

1.2 Group Assignment and Administration Dose

Zebrafish were randomly assigned into 5 groups, the assignment was shown in the table below (table 4):

TABLE 4 Summary of group assignment Group Treatment Blank control (BC) B16-F10-mCherry cells were implanted into zebrafish embryos Vector control (VC) B16-F10-mCherry cells transfected with pcDNA3.1 plasmids were implanted into zebrafish embryos Model control (VEC) B16-F10-mCherry cells transfected with pcDNA3.1 plasmids encoding human VEGFA were implanted into zebrafish embryos EPS1108P-250 μg/ml B16-F10-mCherry cells transfected with EPS1108P-1250 μg/ml pcDNA3.1 plasmids encoding human VEGFA were implanted into zebrafish embryos; and the zebrafish embryos were treated with EPS1108P

10 nanoliters of EPS1108P solution (250, 1250 μg/ml) were injected into the yolk sac of zebrafish embryos by using an electronically regulated air-pressure microinjector at 12 h after tumor cells implanted. Tumor growth and neovascularization in zebrafish embryos were observed and recorded under a laser scanning confocal microscope (Lieca SP5 II) at 12 h after EPS1108P administrated. the areas of tumor vessel and tumor were determined by Image J software, and the ratio of areas (tumor vessel/tumor) were calculated.

1.3 Data Analysis

Data are presented as mean±SD and analyzed by SPSS19.0 software (IBM Corporation). Difference among groups was determined with one-way analysis of variance (ANOVA). Comparison is considered to be statistically significant if p<0.05. When a significant difference is determined, the Least Significant Difference test was performed for further analysis.

2. Result

The ratio of areas (tumor vessel/tumor) in each group is listed in the table below (table 5):

TABLE 5 The ratio of areas (tumor vessel/tumor) in each group ratio of areas Group (tumor vessel/tumor) % Blank control (BC) 28.54 ± 6.61 Vector control (VC) 25.91 ± 5.61 Model control (VEC) 78.79 ± 9.37^(a,b) EPS1108P-250 μg/ml 54.12 ± 1.48^(c) EPS1108P-1250 μg/ml 46.38 ± 2.28^(c) ^(a)Compared with blank control (BC) group, the difference is significant (p < 0.05); ^(b)Compared with Vector control (VC) group, the difference is significant (p < 0.05). ^(c)Compared with Model control (VEC) group, the difference is significant (p < 0.05); 3. Conclusion

Compared with blank control (BC) and vector control (VC), The ratio of areas (tumor vessel/tumor) in model control group (VEC) group was significantly increased (p<0.05), the results indicated that hVEGFA significantly induced tumor neovascularization, the zebrafish xenograft tumor model in zebrafish was established successfully.

Compared with model control (VEC), the ratio of areas (tumor vessel/tumor) in EPS1108P-250 μg/ml and EPS1108P-1250 μg/ml group was significantly decreased (p<0.05), the results indicated that EPS1108P could significantly inhibit the tumor neovascularization induced by human VEGFA.

Example 23: Serum Half-Life (T_(1/2)) In Vivo

1. Experimental Methods and Procedures

1.1 Animal Study

SD rats (Sichuan Dashuo Biotech Inc. SCXK [Sichuan] 2015-030), weight 200-250 g, were randomly assigned into 4 groups, the group assignment and dose information were listed in table below (table 6):

TABLE 6 The group assignment and dose information group animal number pathway dose Volume EPS1108P SD rats, Male 3 i.v 1 mg/kg 4 ml/kg EPS1104P SD rats, Male 3 i.v 1 mg/kg 4 ml/kg EPS1113P SD rats, Male 3 i.v 1 mg/kg 4 ml/kg Aflibercept SD rats, Male 3 i.v 1 mg/kg 4 ml/kg

All test fusion proteins were diluted to 0.25 mg/ml with saline under sterile conditions. A single dose was administrated by i.v. injection (lmg/kg); 300 μL blood were collected from each rat at 5 min, 1 h, 6 h, 24 h, 48 h, 96 h and 144 h post injection via jugular vein. The blood samples were clotted for 1 h at room temperature, then the blood was centrifuged at 1000 g for 15 minutes, serum samples were separated, collected and stored at −80° C. The serum samples were analyzed by ELISA assay.

1.2 Sample Analysis

1.2.1 VEGF₁₆₅ ligand (0.5 μg/ml, R&D Systems, Cat. No. 293-VE) was coated in a 96 well plate, incubated overnight at room temperature;

1.2.2 Washed the plate 3 times with PBST, Blocked plates by adding 300 μL PBS containing 3% BSA, then incubated for 2 h at 37° C.

1.2.3 Washed the plate 3 times with PBST, then added 100 μL of serially diluted standard samples and the serum samples into the wells, and then incubated for 2 h at 37° C.

1.2.4 Washed the plate 3 times with PBST, then added 100 μL of the diluted detection antibody solution (450 ng/ml), then incubated for 2 h at 37° C.

1.2.5 Washed the plate 3 times with PBST, then added 100 μL of the pre-prepared Streptavidin-Horseradish Peroxidase solution, then incubated for 20 min at room temperature.

1.2.6 Washed the plate 3 times with PBST, than added 100 μL of the TMB solution, then incubated for 20 min at room temperature.

1.2.7 Added 504, of the stop solution, gently tap the plate to ensure thorough mixing.

1.2.8 Determined the optical density (OD valume) of each well immediately, using a microplate reader set to 450 nm.

1.2.9 Serum concentration of the test samples were calculated by using the four-parameter fit of the standard curve.

2. Results

The pharmacokinetic parameters (T_(1/2)) were calculated by using the non-compartmental model in DAS3.0 software (Drug and Statistics, Wannan Medical College, Wuhu, China).

TABLE 7 T_(1/2) in each group after single rat i.v injection T_(1/2) Fusion protein X ± SD, h EPS1108P  4.95 ± 0.43 EPS1104P 17.76 ± 3.76 EPS1113P TBD Aflibercept 42.00 ± 6.45 3. Conclusion

The tested fusion proteins of the invention comprised domain of PDGFR and domain of VEGFR which are attached by a linker consisting of proline, alanine and serine, the length (the number of amino acid residues) of the linkers in EPS1108P, EPS1104P and EPS1113P were 200, 400 and 600, respectively. The result indicated that the half-life (T½) of the proteins in rat became longer with increasing length of the amino acid residues. The T½ of EPS1113P was obviously extended.

Example 24: Efficacy of EPS1108P on Inhibition of Laser-Induced Choroidal Neovascularization (CNV) in Cynomolgus Monkeys

1. Establishment of Laser-Induced CNV Model

1.1 CNV Model Induction

Laser photocoagulation was performed on screened animals and laser treatment day was recorded as Day 1 (D1).

Modeling method: Choroidal neovascularization was induced by binocular laser photocoagulations with 6-8 spots in each eye.

Procedure:

1) Mydriasis: Both eyes of an animal were instilled with 1-2 drops of 0.5% Compound Tropicamide Eye Drops.

2) Anesthesia: The animals were anesthetized with Zoletil® intramuscular injection, the absence of corneal reflection, loose in limbs and abdominal muscle and steady breath indicated a moderate anesthesia.

3) Laser photocoagulation: Carbomer Eye Drops (0.2%) were delivered to the eyes before laser photocoagulation, then placed the laser lens appropriately onto the eye to observe the fundus clearly, laser photocoagulation was conducted in the perimacular region which are about 1.5-2PD disk diameter from the foveal center. Care was taken to avoid any visible vessels. The laser parameters were as following: wavelength, 532 nm; power, 400-500 mW; spot size, 50 μm; and exposure time, 100 ms. 4) Animal care: The eyes of animals were smeared with ofloxacin eye ointment after laser photocoagulation. The animals were put on the blanket to keep warm, and put back to the cages after they were conscious. 1.2 Assessment of Success CNV

On Day 14, fluorescein leakage of the laser spots were tested to grade the CNV lesion by fundus fluorescein angiography. Four grades were assigned according to the degree of fluorescein leakage as follow: Grade 1, no hyperfluorescence; Grade 2, hyperfluorescence without leakage; Grade 3, early hyperfluorescence and late mild leakage within the border of fluorescence burn area; Grade 4, early hyperfluorescence and late severe dye leakage beyond the border of the burn area. Meanwhile, the leakage area of Grade 4 lesions will be measured for randomization.

2. Group Assignment

On Day 15, animals with Grade 4 fluorescein leakage lesions were selected for randomization, the average leakage area and rate of Grade 4 lesions were taken into account for group assignment to make sure there was no significance among groups. The specific group assignment is shown in the table below (table 8):

TABLE 8 The Group Assignment Dose Eye Dose Concentration volume number group (μg/eye) (mg/mL) (μL) (N) Vehicle Control — — — 4 Aflibercept-500 μg/Eye 500 10 50 4 EPS1108P-250 μg/Eye 250  5 50 4 EPS1108P-500 μg/Eye 500 10 50 4 3. Dose Procedure Dose Route: Intravitreal injection Dose Frequency and Duration: Single dose on Day 15. Dose Volume: 50 μL/eye, both eyes

Dosing Method: Both eyes of each animal were instilled with 1-2 drops of 0.5% Tropicamide Eye Drops, then anesthesia were conducted as described in CNV Model Induction. Following anesthesia, the animals were laid on an operating table, limbus pal pebralis, eyelash, skin and hair around the eyes were disinfected with povidone iodine. The eyeball was fully exposed, the intravitreal injection was performed at 2-3 mm behind the superior temporal or nasal limbus carefully, toavoid damage to posterior lens capsule and other parts of retina, kept the pinhead in vitreous chamber for 2-5 seconds after injection, then withdrew the needle slowly. After the needle was pulled out, pressed the injection point immediately for about 10 seconds with povidone iodine swabs, ofloxacin eye ointment were applied twice daily for the first three days. The animals were put on the blanket to keep them warm before they get consciousness and put back after they were conscious.

4. Ocular Examination

Before ocular examination, the animals' both eyes were instilled with 1-2 drops of 0.5% Tropicamide Eye Drops, then anesthesia was conducted as described in CNV Model Induction.

4.1 General Ocular Examination

A general ocular examination was conducted. The observation contents of general ocular examinations include eyelid, conjunctiva, cornea, iris, sclera, pupil, lens, vitreous and fundus.

4.2 Fundus Photography and Fluorescein Angiography (FP & FFA)

Fundus photography and fluorescein angiography were conducted on all the animals prior to model induction, and on D14, D22, D29, D36 and D43. Animals were given Fluorescein Sodium Injection (10 mg/kg, 100 mg/mL) by intravenous injection before fluorescein angiography.

Observation: Compared the early and late phase FFA images to detect and measure the evidence and extent of leakage of CNV. If CNV is present, hyperfluorescence develops around the laser spot, which progresses to late diffuse leakage with dye pooling in the serous detachment surrounding the burn area. The leakage was graded on a standardized scale of 1 to 4; grading scores were defined in Section Assessment of Successful CNV. Grade 4 lesions was defined as clinically significant fluorescence leakage of classic experimental CNV model, the area of leakage was measured. Meanwhile, the rate of Grade 4 lesions in each group was calculated by following formulas: Rate of Grade 4 lesions (%)=number of Grade 4 lesions/number of the laser spots*100% 5. Statistical Analysis

Data are presented as mean±SD and analyzed by SPSS13.0 software (IBM Corporation). Difference among mean of the groups is determined with ANOVA. Comparison is considered to be statistically significant if p<0.05. When a significant difference is determined, the Least Significant Difference test was performed for further analysis. In the case of heterogeneity of variance at p<0.05, a Kruskal-Wallis test was performed.

6. Result

No abnormalities were found on the fundus photography or FFA in any eye of these animals before the CNV induction. After Laser induction, Fundus photography and FFA were performed on D14, D22, D29, D36 and D43; no evidence of fundus abnormalities except laser photocoagulation lesions was found on the fundus photography.

6.1.1 The Rate of Grade 4 Lesion Occurrence

Summary of Grade 4 lesion rate is shown in the table below (Table 9):

TABLE 9 Summary of Grade 4 lesion rate in each group pre/post dosing Grade 4 lesion rate (%) D14 D22 (7 d D29 (14 d D36 (21 d D43 (28 d Group (pre-dose) post-dose) post-dose) post-dose) post-dose) Vehicle Control Mean ± SD 75.4 ± 14.2 64.7 ± 19.1 60.7 ± 31.7 64.3 ± 27.3 64.3 ± 27.3 n 4 4 4 4 4 Aflibercept- Mean ± SD 75.0 ± 41.0 10.7 ± 21.5 ^(a)   3.6 ± 7.2 ^(a)    7.2 ± 14.3 ^(a)   14.3 ± 28.6 ^(a) 500 μg/Eye n 4 4 4 4 4 EPS1108P- Mean ± SD 75.0 ± 24.4   53.6 ± 17.9 ^(b) 25.0 ± 18.0   17.9 ± 18.0 ^(a)   17.9 ± 18.0 ^(a) 250 μg/Eye n 4 4 4 4 4 EPS1108P- Mean ± SD 74.4 ± 31.4 38.1 ± 27.5   18.5 ± 26.9 ^(a)   14.9 ± 20.3 ^(a)   18.5 ± 26.9 ^(a) 500 μg/Eye n 4 4 4 4 4 n: the number of Eyes; ^(a) Compared with Vehicle Control group, the difference is significant ( p <0.05); ^(b) Compared with Aflibercept-500 μg/Eye group, the difference is significant (p <0.05). 6.1.2 The Average Area of Leakage

Summary of the average area of fluorescein leakage is shown in the table below (Table 10):

TABLE 10 The average area of fluorescein leakage in each group pre/post dosing Average area of fluorescein leakage (mm²) D14 D22 (7 d D29 (14 d D36 (21 d D43 (28 d Group (pre-dose) post-dose) post-dose) post-dose) post-dose) Vehicle Control Mean ± SD 1.56 ± 0.61 1.57 ± 0.73 1.77 ± 1.07 1.86 ± 1.01 1.92 ± 1.05 n 4 4 4 4 4 Aflibercept- Mean ± SD 1.51 ± 0.61   0.61 ± 0.30 ^(a)   0.52 ± 0.33 ^(a)   0.54 ± 0.45 ^(a) 0.64 ± 0.38 500 μg/Eye n 4 4 4 4 4 EPS1108P- Mean ± SD 1.54 ± 0.46 0.94 ± 0.50 0.88 ± 0.49 1.06 ± 1.02 1.04 ± 0.97 250 μg/Eye n 4 4 4 4 4 EPS1108P- Mean ± SD 1.52 ± 0.47   0.75 ± 0.16 ^(a)   0.68 ± 0.35 ^(a)   0.71 ± 0.39 ^(a) 0.77 ± 0.30 500 μg/Eye n 4 4 4 4 4 n: the number of Eyes; ^(a) Compared with Vehicle Control group, the difference is significant ( p <0.05); ^(b) Compared with Aflibercept-500 μg/Eye group, the difference is significant (p <0.05). 7. Conclusion

The Fundus photography and fluorescein angiography (FP & FFA) results indicated that the animal eyelaser-induced CNV Model was successfully established. When the animals were treated with EPS1108P (250 and 500 μg/Eye) by single Intravitreal injection (IVT), the Grade 4 lesion rate and the average area of fluorescein leakage were significantly decreased, and the inhibition effect was dose dependent, which showed that EPS1108P is effective drug in treating CNV in the cynomolgus monkey model.

Compared with the positive control, the inhibitory effects of EPS1108P were comparable to that of aflibercept at D36 (21 d post-dose) and D43 (28 d post-dose); the positive control immediately improved the Grade 4 lesion rate and the average area of fluorescein leakage, while EPS1108P was more gentle, and sustainably inhibited to the same level as the positive control.

Example 25: Pharmacokinetic Study of Single Intravitreal Injection in New Zealand Rabbits

1. Experimental Methods and Procedures

1.1 Animal Study

New Zealand rabbit, 2-2.5 kg, male or female, purchased from Chengdu Dashuo Experimental Animal Co., Ltd. (license No. SCXK [Sichuan] 2015-030). All rabbits were randomly divided into 3 groups; the group and dose information are listed in the table below (Table 11):

TABLE 11 The group and dose information group number pathway dose volume EPS1108P 3 intravitreal 250 μg/eye 50 μL/eye injection EPS1104P 3 intravitreal 500 μg/eye 50 μL/eye injection Aflibercept 3 intravitreal 500 μg/eye 50 μL/eye injection

All test fusion proteins were diluted with saline under sterile conditions.

All animals were allowed to acclimatize for at least 7 days prior to experiments. Before administration, 2 drops of oxybuprocaine hydrochloride eye drops (#B2030, Santen Pharmaceutical CO., Ltd.) were dripped into the rabbit eyes and wiped with 5% povidone iodine on the periocular region and the conjunctiva of the eyes. A single dose of the different test fusion proteins was administrated by intravitreal injection (50 μL/eye) after eye local anesthesia. The eyes of rabbits were excised at each of the following time points: Day 1, 4, 8, 12, 16 and 21 days after injection. The vitreous was collected and immediately frozen at −80° C. The vitreous samples were analyzed by ELISA assay.

2. Results

The pharmacokinetic parameters (T_(1/2)) were calculated by using the non-compartmental model in Phoenix.

TABLE 12 The estimated half-life (T_(1/2)) of each group Fusion protein T_(1/2) (Day) EPS1108P 5.77 EPS1104P 8.72 Aflibercept 4.26 3. Conclusion

The tested fusion proteins of the invention comprised domains of PDGFR and domains of VEGFR which were attached by a linker consisting of proline, alanine and serine, wherein the length (i.e., the number of amino acid residues) of the linkers in EPS1108P and EPS1104P were 200 and 400, respectively.

The result indicated that the half-life (T½) of the proteins in New Zealand rabbits became longer with increasing length of the linker. Compared with Aflibercept, whose reported half life was 3.9 days (Park S J, Choi Y, Na Y M, et al. Intraocular pharmacokinetics of intravitreal aflibercept (eylea) in a rabbit model. Invest Ophthalmol Vis Sci. 2016; 57:2612-2617.), the T_(1/2) of both EPS1108P and EPS1104P was significantly longer. A significantly longer half-life means the potential to be a longer-acting drug, which can significantly reduce the frequency of administration of ophthalmic patients, reduce the risk of eye infections, and reduce the pain and financial burden of patients.

Example 26: Native PAGE and Electromobility Gel Shift Assay

EPS1104P was mixed with VEGF₁₆₅ (#C083, Novoprotein, Shanghai, China), PDGF-BB (#C199, Novoprotein, Shanghai, China) and VEGF+PDGF-BB, and incubated in an ice bath for 30 min. 40 μL of the above three incubation mixtures and EPS1104P were added to 10 μl of 5× Loading buffer (#ES005, Wanshenghaotian, Shanghai, China), respectively, and the four samples were loaded on a native PAGE gel (#NGSH2001-8T, Wanshenghaotian, Shanghai, China). The electrophoresis was run at 70 V for 6 hours. The gel was stained with Coomassie blue and then bleached. The gel electropherogram is shown in FIG. 16 . The electropherogram reveals that the molecular weights of lanes 2 (EPS1104P+PDGF-BB), 3 (EPS1104P+VEGF₁₆₅), and 4 (EPS1104P+VEGF₁₆₅+PDGF-BB) are larger than those of lane 1 (EPS1104P), indicating that EPS1104P can be combined with VEGF₁₆₅ or PDGF-BB alone to each form a stable complex. It is also possible to combine EPS1104P with both VEGF165 and PDGF-BB to form a stable complex.

Example 27: Inhibition of VEGF₁₆₅-Induced HUVEC Proliferation

1. Assay Methods

1.1 A blank control group, a VEGF control group, and a test sample group (EPS1104P) were established in the experiment. Three parallel wells were set in each group, and the experiment was repeated three times.

1.2 HUVEC cells growing in an exponential growth phase were harvested and prepared for single cell suspension.

1.3 The cells were counted and adjusted to a density of 5×10⁴ cells/mL with basal Medium (#1001-b, Sciencell). 100 μL of the cell suspension was seeded into a 96 well plate. Incubated at 37° C., 5% CO₂ overnight (without feed).

1.4 The dilution medium was mixed with a complete medium (#1001, Sciencell) and a basal medium. 100 μl of dilution medium without VEGF₁₆₅ was added to the well of the blank control group. 100 μl of dilution medium containing 25 ng/ml of VEGF₁₆₅ was added to the well of the VEGF control group. EPS1104P was serially diluted to working concentrations (200 nM, 50 nM, 12.5 nM, 3.125 nM, 0.781 nM, 0.195 nM, 0.049 nM and 0.012 nM) with dilution medium containing 25 ng/ml of VEGF₁₆₅0.100 μL of the diluted EPS1104P was added to the well of the test sample group. The 96 well plate of the three groups was incubated for 72 h at 37° C., 5% CO₂. 1.5 After incubation, 20 μL of Cell Counting Kit-8 (#CK04, Dojindo, Shanghai, China) was added into each well, then incubated in incubator for 2-3 h. 1.6 Absorbance (OD value) was measured at 450 nm by using a microplate reader (Thermofisher). 1.7 The IC₅₀ in each group was calculated with Origin. 2. Results

TABLE 13 Inhibition of HUVEC cells proliferation Sample IC₅₀ (nM) EPS1104P 1.43 3. Conclusion

EPS1104P showed significant inhibition in the VEGF₁₆₅-induced HUVEC cell proliferation test.

The present disclosure refers to the following nucleotide and amino acid sequences.

Some of the sequences provided herein are, inter alia, available in the NCBI database and can be retrieved from www.ncbi.nlm.nih.gov/sites/entrez?db=gene; Theses sequences also relate to annotated and modified sequences. Techniques and methods are provided herein wherein homologous sequences, and variants of the concise sequences provided herein are used. Preferably, such “variants” are genetic variants.

SEQ ID No. 1: Nucleotide sequence encoding PAS linker Gcctctcctgctgcccctgccccagcttctccagctgctcctgcaccttctgctccagccgctagtcctgcagctccagctcc tgcttctcctgccgcaccagcacctagtgcccctgctgcatcaccagcagctcccgcacccgctagcccagctgcaccagctc caagtgctccagcagcttcacccgcagcacccgctccagcaagtccagcagccccagccccttcagcaccagctgcatctccc gcagcccctgctcctgccagccctgccgctcctgctccaagcgctcctgctgctagtccagccgcccctgcaccagcaagtcc tgctgctcccgcacctagtgcaccagcagcaagccctgcagctcctgcaccagcatctccagcagcaccagcaccatcagccc ctgccgcttctcccgcagctccagccccagcctcccctgctgctccagccccctctgctcctgcagcatctcctgccgctccc gcccctgcaagtcccgccgctccagcaccatccgctccagctgcttccccagccgctccagctccagctagccccgcagcccc cgcaccatctgccccagca SEQ ID No. 2: Amino acid sequence of PAS linker ASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASP AAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAP APASPAAPAPSAPAASPAAPAPASPAAPAPSAPA SEQ ID No. 3: Nucleotide sequence encoding Ig domains 1 to 3 of PDGFRα cagctgagcctgccctccatcctgcctaacgagaatgagaaggtggtgcagctgaactccagcttctccctgagatgctttgg cgagtctgaggtgtcctggcagtacccaatgagcgaggaggagtcttccgacgtggagatccgcaatgaggagaacaattctg gcctgttcgtgaccgtgctggaggtgagctctgcctccgccgctcacaccggcctgtacacatgttactataaccatacccag acagaggagaatgagctggagggcagacacatctacatctatgtgcccgatcctgacgtggcctttgtgccactgggcatgac cgattacctggtcatcgtggaggacgatgacagcgccatcatcccctgcaggaccacagaccccgagacacctgtgacactgc ataactctgagggcgtggtgccagccagctacgattctcggcagggcttcaatggcacctttacagtgggcccctatatctgt gaggccaccgtgaagggcaagaagttccagacaatcccttttaacgtgtacgccctgaaggctaccagcgagctggacctgga gatggaggccctgaagacagtgtataagtctggcgagacaatcgtggtgacatgcgccgtgttcaacaatgaggtggtggatc tgcagtggacctaccccggcgaggtgaagggcaagggcatcacaatgctggaggagatcaaggtgccttctatcaagctggtg tacaccctgacagtgccagaggccaccgtgaaggattccggcgactatgagtgtgccgctaggcaggctacccgggaggtgaa ggagatgaagaaggtgacaatctctgtgcacgagaaggga SEQ ID No. 4: Amino acid sequence of Ig domains 1 to 3 of PDGFRα QLSLPSILPNENEKVVQLNSSFSLRCFGESEVSWQYPMSEEESSDVEIRNEENNSGLFVTVLEVSSASAAHTGLYTCYYNHTQ TEENELEGRHIYIYVPDPDVAFVPLGMTDYLVIVEDDDSAIIPCRTTDPETPVTLHNSEGVVPASYDSRQGFNGTFTVGPYIC EATVKGKKFQTIPFNVYALKATSELDLEMEALKTVYKSGETIVVTCAVFNNEVVDLQWTYPGEVKGKGITMLEEIKVPSIKLV YTLTVPEATVKDSGDYECAARQATREVKEMKKVTISVHEKG SEQ ID No. 5: Nucleotide sequence encoding Ig domains 1 to 3 of PDGFRβ aacgatgccgaggaactgttcatcttcctgaccgagattaccgagatcacaatcccctgccgcgtgacagatcctcagctggt ggttaccctgcatgagaagaaaggcgacgtggccctgcctgtgccttacgatcatcagagaggcttctccggcatcttcgagg accggtcttacatctgcaagaccaccatcggcgacagagaggtggactccgacgcctactacgtgtacagactccaggtgtcc tccatcaacgtgtccgtgaatgccgtgcagacagttgtgcggcagggcgagaatatcaccctgatgtgcatcgtgatcggcaa cgaggtggtcaacttcgagtggacctatcctcggaaagaatctggccggctggtggaacctgtgaccgacttcctgctggaca tgccctaccacatccggtctatcctgcacatcccttccgccgagctggaagattccggcacctacacctgtaacgtgaccgag tccgtgaacgaccaccaggacgagaaggccatcaatatcaccgtggtggaatccggctacgtgcggctgttgggagaagtggg cacactgcagtttgctgagctg SEQ ID No. 6: Amino acid sequence of Ig domains 1 to 3 of PDGFRβ NDAEELFIFLTEITEITIPCRVTDPQLVVTLHEKKGDVALPVPYDHQRGFSGIFEDRSYICKTTIGDREVDSDAYYVYRLQVS SINVSVNAVQTVVRQGENITLMCIVIGNEVVNFEWTYPRKESGRLVEPVTDFLLDMPYHIRSILHIPSAELEDSGTYTCNVTE SVNDHQDEKAINITVVESGYVRLLGEVGTLQFAEL SEQ ID No. 7: Nucleotide sequence encoding Ig domain 2 of VEGFR-1 and Ig domain 3 of VEGFR-2 agtgataccggtagacctttcgtagagatgtacagtgaaatccccgaaattatacacatgactgaaggaagggagctcgtcat tccctgccgggttacgtcacctaacatcactgttactttaaaaaagtttccacttgacactttgatccctgatggaaaacgca taatctgggacagtagaaagggcttcatcatatcaaatgcaacgtacaaagaaatagggcttctgacctgtgaagcaacagtc aatgggcatttgtataagacaaactatctcacacatcgacaaaccaatacaatcatagatgtggttctgagtccgtctcatgg aattgaactatctgttggagaaaagctcgtcttaaattgtacagcaagaactgaactaaatgtggggattgacttcaactggg aatacccttcttcgaagcatcagcataagaaacttgtaaaccgagacctaaaaacccagtctgggagtgagatgaagaaattt ttgagcaccttaactatagatggtgtaacccggagtgaccaaggattgtacacctgtgcagcatccagtgggctgatgaccaa gaagaacagcacatttgtcagggtccatgaaaag SEQ ID No. 8: Amino acid sequence of Ig domain 2 of VEGFR-1 and Ig domain 3 of VEGFR-2 SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATV NGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKF LSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK SEQ ID No. 9: Nucleotide sequence encoding N-terminal signal polypeptide sequence atggggacttcccatccggcgttcctggtcttaggctgtcttctcacagggctgagcctaatcctctgc SEQ ID No. 10: Amino acid sequence of N-terminal signal polypeptide sequence MGTSHPAFLVLGCLLTGLSLILC SEQ ID No. 11: Nucleotide sequence encoding purification tag caccatcaccatcaccacgcc SEQ ID No. 12: Amino acid sequence of purification tag HHHHHHA SEQ ID No. 13: Nuleotide sequence of vector pDSG33-PDGFR-PAS200-VEGFR (7190 bp) acccacaatggggacttcccatccggcgttcctggtcttaggctgtcttctcacagggctgagcctaatcctctgccagc tttcattaccctctatccttccaaatgaaaatgaaaaggttgtgcagctgaattcatccttttctctgagatgctttggg gagagtgaagtgagctggcagtaccccatgtctgaagaagagtcttccgatgtggaaatcagaaatgaagaaaacaacag cggcctttttgtgacggtcttggaagtgagcagtgcctcggcggcccacacagggttgtacacttgctattacaaccaca ctcagacagaagagaatgagcttgaaggcaggcacatttacatctatgtgccagacccagatgtagcctttgtacctcta ggaatgacggattatttagtcatcgtggaggatgatgattctgccattataccttgtcgcacaactgatcccgagactcc tgtaaccttacacaacagtgagggggtggtacctgcctcctacgacagcagacagggctttaatgggaccttcactgtag ggccctatatctgtgaggccaccgtcaaaggaaagaagttccagaccatcccatttaatgtttatgctttaaaagcaaca tcagagctggatctagaaatggaagctcttaaaaccgtgtataagtcaggggaaacgattgtggtcacctgtgctgtttt taacaatgaggtggttgaccttcaatggacttaccctggagaagtgaaaggcaaaggcatcacaatgctggaagaaatca aagtcccatccatcaaattggtgtacactttgacggtccccgaggccacggtgaaagacagtggagattacgaatgtgct gcccgccaggctaccagggaggtcaaagaaatgaagaaagtcactatttctgtccatgagaaaggtgcctctcctgctgc ccctgccccagcttctccagctgctcctgcaccttctgctccagccgctagtcctgcagctccagctcctgcttctcctg ccgcaccagcacctagtgcccctgctgcatcaccagcagctcccgcacccgctagcccagctgcaccagctccaagtgct ccagcagcttcacccgcagcacccgctccagcaagtccagcagccccagccccttcagcaccagctgcatctcccgcagc ccctgctcctgccagccctgccgctcctgctccaagcgctcctgctgctagtccagccgcccctgcaccagcaagtcctg ctgctcccgcacctagtgcaccagcagcaagccctgcagctcctgcaccagcatctccagcagcaccagcaccatcagcc cctgccgcttctcccgcagctccagccccagcctcccctgctgctccagccccctctgctcctgcagcatctcctgccgc tcccgcccctgcaagtcccgccgctccagcaccatccgctccagctgcttccccagccgctccagctccagctagccccg cagcccccgcaccatctgccccagcagccagtgataccggtagacctttcgtagagatgtacagtgaaatccccgaaatt atacacatgactgaaggaagggagctcgtcattccctgccgggttacgtcacctaacatcactgttactttaaaaaagtt tccacttgacactttgatccctgatggaaaacgcataatctgggacagtagaaagggcttcatcatatcaaatgcaacgt acaaagaaatagggcttctgacctgtgaagcaacagtcaatgggcatttgtataagacaaactatctcacacatcgacaa accaatacaatcatagatgtggttctgagtccgtctcatggaattgaactatctgttggagaaaagctcgtcttaaattg tacagcaagaactgaactaaatgtggggattgacttcaactgggaatacccttcttcgaagcatcagcataagaaacttg taaaccgagacctaaaaacccagtctgggagtgagatgaagaaatttttgagcaccttaactatagatggtgtaacccgg agtgaccaaggattgtacacctgtgcagcatccagtgggctgatgaccaagaagaacagcacatttgtcagggtccatga aaagcaccatcaccatcaccacgcctgaagagcttaagcttgcggccgcagatctagcttaagtttaaaccgctgatcag cctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccact cccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggt ggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggagcttggcc gcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaa cccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgctta ccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtg taggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcg tcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatg taggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctg ctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttt tgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctc agtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaa aaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacc tatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggct taccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagcca gccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctag agtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttg gtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagc tccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattc tcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgc ggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcatt ggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacc caactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagg gaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgt ctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgcc acctgacgtctaggttcacctaagaatgggagcaaccagcaggaaaaggacaagcagcgaaaattcacgcccccttggga ggtggcggcatatgcaaaggatagcactcccactctactactgggtatcatatgctgactgtatatgcatgaggatagca tatgctacccggatacagattaggatagcatatactacccagatatagattaggatagcatatgctacccagatatagat taggatagcctatgctacccagatataaattaggatagcatatactacccagatatagattaggatagcatatgctaccc agatatagattaggatagcctatgctacccagatatagattaggatagcatatgctacccagatatagattaggatagca tatgctatccagatatttgggtagtatatgctacccagatataaattaggatagcatatactaccctaatctctattagg atagcatatgctacccggatacagattaggatagcatatactacccagatatagattaggatagcatatgctacccagat atagattaggatagcctatgctacccagatataaattaggatagcatatactacccagatatagattaggatagcatatg ctacccagatatagattaggatagcctatgctacccagatatagattaggatagcatatgctatccagatatttgggtag tatatgctacccatggcaacattagcccaccgtgctctcagcgacctcgtgaatatgaggaccaacaaccctgtgcttgg cgctcaggcgcaagtgtgtgtaatttgtcctccagatcgcagcaatcgcgcccctatcttggcccgcccacctacttatg caggtattccccggggtgccattagtggttttgtgggcaagtggtttgaccgcagtggttagcggggttacaatcagcca agttattacacccttattttacagtccaaaaccgcagggcggcgtgtgggggctgacgcgtgcccccactccacaatttc aaaaaaaagagtggccacttgtctttgtttatgggccccattggcgtggagccccgtttaattttcgggggtgttagaga caaccagtggagtccgctgctgtcggcgtccactctctttccccttgttacaaatagagtgtaacaacatggttcacctg tcttggtccctgcctgggacacatcttaataaccccagtatcatattgcactaggattatgtgttgcccatagccataaa ttcgtgtgagatggacatccagtctttacggcttgtccccaccccatggatttctattgttaaagatattcagaatgttt cattcctacactagtatttattgcccaaggggtttgtgagggttatattggtgtcatagcacaatgccaccactgaaccc cccgtccaaattttattctgggggcgtcacctgaaaccttgttttcgagcacctcacatacaccttactgttcacaactc agcagttattctattagctaaacgaaggagaatgaagaagcaggcgaagattcaggagagttcactgcccgctccttgat cttcagccactgcccttgtgactaaaatggttcactaccctcgtggaatcctgaccccatgtaaataaaaccgtgacagc tcatggggtgggagatatcgctgttccttaggacccttttactaaccctaattcgatagcatatgcttcccgttgggtaa catatgctattgaattagggttagtctggatagtatatactactacccgggaagcatatgctacccgtttagggttaaca agggggccttataaacactattgctaatgccctcttgagggtccgcttatcggtagctacacaggcccctctgattgacg ttggtgtagcctcccgtagtcttcctgggcccctgggaggtacatgtcccccagcattggtgtaagagcttcagccaaga gttacacataaaggcaatgttgtgttgcagtccacagactgcaaagtctgctccaggatgaaagccactcagtgttggca aatgtgcacatccatttataaggatgtcaactacagtcagagaacccctttgtgtttggtccccccccgtgtcacatgtg gaacagggcccagttggcaagttgtaccaaccaactgaagggattacatgcactgccccgcattaattgcatgaagaatc tgcttagggttaggcgttttgcgctgcttcgcgatgtacgggccagatatacgcgttgacattgattattgactagttat taatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggccc gcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactt tccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacg ccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttg gcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggt ttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggacttt ccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagc tctctggctaactagagaacccactgcttactggcttatcgaaattaatacgactcactatagggtctag SEQ ID No. 14: Translation of pDSG33-PDGFR-PAS200-VEGFR nucleotides 8-2188 coding for protein sequence PDGFR_(αD123)-PAS(200)-VEGFR1_(D)2/R2_(D3) (726 amino acids; includes signal sequence and purification tag) MGTSHPAFLVLGCLLTGLSLILCQLSLPSILPNENEKVVQLNSSFSLRCFGESEVSWQYPMSEEESSDVEIRNEENNSGL FVTVLEVSSASAAHTGLYTCYYNHTQTEENELEGRHIYIYVPDPDVAFVPLGMTDYLVIVEDDDSAIIPCRTTDPETPVT LHNSEGVVPASYDSRQGFNGTFTVGPYICEATVKGKKFQTIPFNVYALKATSELDLEMEALKTVYKSGETIVVTCAVFNN EVVDLQWTYPGEVKGKGITMLEEIKVPSIKLVYTLTVPEATVKDSGDYECAARQATREVKEMKKVTISVHEKGASPAAPA PASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPA PASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPA PASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPL DTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTA RTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKH HHHHHA SEQ ID No. 15: Nucleotide sequence encoding EPS1108P excluding signal polypeptide sequence and purification-tag cagctttcattaccctctatccttccaaatgaaaatgaaaaggttgtgcagctgaattcatccttttctctgagatgctttgg ggagagtgaagtgagctggcagtaccccatgtctgaagaagagtcttccgatgtggaaatcagaaatgaagaaaacaacagcg gcctttttgtgacggtcttggaagtgagcagtgcctcggcggcccacacagggttgtacacttgctattacaaccacactcag acagaagagaatgagcttgaaggcaggcacatttacatctatgtgccagacccagatgtagcctttgtacctctaggaatgac ggattatttagtcatcgtggaggatgatgattctgccattataccttgtcgcacaactgatcccgagactcctgtaaccttac acaacagtgagggggtggtacctgcctcctacgacagcagacagggctttaatgggaccttcactgtagggccctatatctgt gaggccaccgtcaaaggaaagaagttccagaccatcccatttaatgtttatgctttaaaagcaacatcagagctggatctaga aatggaagctcttaaaaccgtgtataagtcaggggaaacgattgtggtcacctgtgctgtttttaacaatgaggtggttgacc ttcaatggacttaccctggagaagtgaaaggcaaaggcatcacaatgctggaagaaatcaaagtcccatccatcaaattggtg tacactttgacggtccccgaggccacggtgaaagacagtggagattacgaatgtgctgcccgccaggctaccagggaggtcaa agaaatgaagaaagtcactatttctgtccatgagaaaggtgcctctcctgctgcccctgccccagcttctccagctgctcctg caccttctgctccagccgctagtcctgcagctccagctcctgcttctcctgccgcaccagcacctagtgcccctgctgcatca ccagcagctcccgcacccgctagcccagctgcaccagctccaagtgctccagcagcttcacccgcagcacccgctccagcaag tccagcagccccagccccttcagcaccagctgcatctcccgcagcccctgctcctgccagccctgccgctcctgctccaagcg ctcctgctgctagtccagccgcccctgcaccagcaagtcctgctgctcccgcacctagtgcaccagcagcaagccctgcagct cctgcaccagcatctccagcagcaccagcaccatcagcccctgccgcttctcccgcagctccagccccagcctcccctgctgc tccagccccctctgctcctgcagcatctcctgccgctcccgcccctgcaagtcccgccgctccagcaccatccgctccagctg cttccccagccgctccagctccagctagccccgcagcccccgcaccatctgccccagcagccagtgataccggtagacctttc gtagagatgtacagtgaaatccccgaaattatacacatgactgaaggaagggagctcgtcattccctgccgggttacgtcacc taacatcactgttactttaaaaaagtttccacttgacactttgatccctgatggaaaacgcataatctgggacagtagaaagg gcttcatcatatcaaatgcaacgtacaaagaaatagggcttctgacctgtgaagcaacagtcaatgggcatttgtataagaca aactatctcacacatcgacaaaccaatacaatcatagatgtggttctgagtccgtctcatggaattgaactatctgttggaga aaagctcgtcttaaattgtacagcaagaactgaactaaatgtggggattgacttcaactgggaatacccttcttcgaagcatc agcataagaaacttgtaaaccgagacctaaaaacccagtctgggagtgagatgaagaaatttttgagcaccttaactatagat ggtgtaacccggagtgaccaaggattgtacacctgtgcagcatccagtgggctgatgaccaagaagaacagcacatttgtcag ggtccatgaaaag SEQ ID No. 16: Amino acid sequence of EPS1108P excluding signal polypeptide sequence and purification-tag QLSLPSILPNENEKVVQLNSSFSLRCFGESEVSWQYPMSEEESSDVEIRNEENNSGLFVTVLEVSSASAAHTGLYTCYYNHTQ TEENELEGRHIYIYVPDPDVAFVPLGMTDYLVIVEDDDSAIIPCRTTDPETPVTLHNSEGVVPASYDSRQGFNGTFTVGPYIC EATVKGKKFQTIPFNVYALKATSELDLEMEALKTVYKSGETIVVTCAVFNNEVVDLQWTYPGEVKGKGITMLEEIKVPSIKLV YTLTVPEATVKDSGDYECAARQATREVKEMKKVTISVHEKGASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAAS PAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAA PAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASDTGRPF VEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKT NYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTID GVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK SEQ ID No. 17: DNA-Sequence of PDGFR_(αD123)-cloning site-VEGFR1_(D)2/R2_(D3) for cloning into pDSG33- PDGFR-PAS200-VEGFR (sequence flanked by restriction sites for XbaI and HindIII; underlined) tctagacccacaatggggacttcccatccggcgttcctggtcttaggctgtcttctcacagggctgagcctaatcctctg ccagctttcattaccctctatccttccaaatgaaaatgaaaaggttgtgcagctgaattcatccttttctctgagatgct ttggggagagtgaagtgagctggcagtaccccatgtctgaagaagagtcttccgatgtggaaatcagaaatgaagaaaac aacagcggcctttttgtgacggtcttggaagtgagcagtgcctcggcggcccacacagggttgtacacttgctattacaa ccacactcagacagaagagaatgagcttgaaggcaggcacatttacatctatgtgccagacccagatgtagcctttgtac ctctaggaatgacggattatttagtcatcgtggaggatgatgattctgccattataccttgtcgcacaactgatcccgag actcctgtaaccttacacaacagtgagggggtggtacctgcctcctacgacagcagacagggctttaatgggaccttcac tgtagggccctatatctgtgaggccaccgtcaaaggaaagaagttccagaccatcccatttaatgtttatgctttaaaag caacatcagagctggatctagaaatggaagctcttaaaaccgtgtataagtcaggggaaacgattgtggtcacctgtgct gtttttaacaatgaggtggttgaccttcaatggacttaccctggagaagtgaaaggcaaaggcatcacaatgctggaaga aatcaaagtcccatccatcaaattggtgtacactttgacggtccccgaggccacggtgaaagacagtggagattacgaat gtgctgcccgccaggctaccagggaggtcaaagaaatgaagaaagtcactatttctgtccatgagaaaggtgccagaaga gcagatctgggctcttctgcccaccatcaccatcaccattaagcttgcggctcttctgccagtgataccggtagaccttt cgtagagatgtacagtgaaatccccgaaattatacacatgactgaaggaagggagctctaagctt SEQ ID No. 18: DNA-Sequence of PDGFR_(αD123)-PAS(200)-VEGFR1_(D)2/R2_(D3) in pDSG33-PDGFR-PAS200- VEGFR (sequence flanked by restriction sites for XbaI and HindIII; underlined) tctagacccacaatggggacttcccatccggcgttcctggtcttaggctgtcttctcacagggctgagcctaatcctctg ccagctttcattaccctctatccttccaaatgaaaatgaaaaggttgtgcagctgaattcatccttttctctgagatgct ttggggagagtgaagtgagctggcagtaccccatgtctgaagaagagtcttccgatgtggaaatcagaaatgaagaaaac aacagcggcctttttgtgacggtcttggaagtgagcagtgcctcggcggcccacacagggttgtacacttgctattacaa ccacactcagacagaagagaatgagcttgaaggcaggcacatttacatctatgtgccagacccagatgtagcctttgtac ctctaggaatgacggattatttagtcatcgtggaggatgatgattctgccattataccttgtcgcacaactgatcccgag actcctgtaaccttacacaacagtgagggggtggtacctgcctcctacgacagcagacagggctttaatgggaccttcac tgtagggccctatatctgtgaggccaccgtcaaaggaaagaagttccagaccatcccatttaatgtttatgctttaaaag caacatcagagctggatctagaaatggaagctcttaaaaccgtgtataagtcaggggaaacgattgtggtcacctgtgct gtttttaacaatgaggtggttgaccttcaatggacttaccctggagaagtgaaaggcaaaggcatcacaatgctggaaga aatcaaagtcccatccatcaaattggtgtacactttgacggtccccgaggccacggtgaaagacagtggagattacgaat gtgctgcccgccaggctaccagggaggtcaaagaaatgaagaaagtcactatttctgtccatgagaaaggtgcctctcct gctgcccctgccccagcttctccagctgctcctgcaccttctgctccagccgctagtcctgcagctccagctcctgcttc tcctgccgcaccagcacctagtgcccctgctgcatcaccagcagctcccgcacccgctagcccagctgcaccagctccaa gtgctccagcagcttcacccgcagcacccgctccagcaagtccagcagccccagccccttcagcaccagctgcatctccc gcagcccctgctcctgccagccctgccgctcctgctccaagcgctcctgctgctagtccagccgcccctgcaccagcaag tcctgctgctcccgcacctagtgcaccagcagcaagccctgcagctcctgcaccagcatctccagcagcaccagcaccat cagcccctgccgcttctcccgcagctccagccccagcctcccctgctgctccagccccctctgctcctgcagcatctcct gccgctcccgcccctgcaagtcccgccgctccagcaccatccgctccagctgcttccccagccgctccagctccagctag ccccgcagcccccgcaccatctgccccagcagccagtgataccggtagacctttcgtagagatgtacagtgaaatccccg aaattatacacatgactgaaggaagggagctcgtcattccctgccgggttacgtcacctaacatcactgttactttaaaa aagtttccacttgacactttgatccctgatggaaaacgcataatctgggacagtagaaagggcttcatcatatcaaatgc aacgtacaaagaaatagggcttctgacctgtgaagcaacagtcaatgggcatttgtataagacaaactatctcacacatc gacaaaccaatacaatcatagatgtggttctgagtccgtctcatggaattgaactatctgttggagaaaagctcgtctta aattgtacagcaagaactgaactaaatgtggggattgacttcaactgggaatacccttcttcgaagcatcagcataagaa acttgtaaaccgagacctaaaaacccagtctgggagtgagatgaagaaatttttgagcaccttaactatagatggtgtaa cccggagtgaccaaggattgtacacctgtgcagcatccagtgggctgatgaccaagaagaacagcacatttgtcagggtc catgaaaagcaccatcaccatcaccacgcctgaagagcttaagctt SEQ ID No. 19: Nucleotide sequence encoding Ig domains 1 to 3 of mutantPDGFR α cagctgagcctgccaagcatcctgcctaacgaaaatgagaaggtggtccagctgaacagctccttcagtctgagatgctttgg cgaatcagaggtgagctggcagtacccaatgtcagaggaagagtctagtgacgtggaaattaggaatgaagagaacaattcag gactgttcgtgaccgtcctggaggtgtcaagcgccagcgccgctcacaccggactgtacacatgttactataaccatactcag accgaagagaatgaactggaggggaggcacatctccatccacgtgcccgatcctgacgtggcctttgccccactgggaatgac agattacctggtcatcgtcgaggacgatgactctgccatcattccctgccgcacctcagactccgaaactcctgtgaccctgc ataacagtgagggcgtggtccccgcctcctacgattctcgacagggattcaatggcaccttcaccgtcggaccctatatctgt gaggccactgtgaagggcaagaaattccagaccattccttttaacgtgtacgcactgaaagccacatccgaactggacctgga aatggaggccctgaagactgtctataaatctggagagactatcgtggtcacctgcgccgtgttcaacaatgaagtggtcgatg cgcagtggacttaccccggcgaggtcaagggcaaagggattaccatggacgaagagatcaaggtgcctagccagaagctggtg tacaccctgacagtcccagaagccaccgtgaaggattccggggactatgagtgtgcagcccggcaggcctccagagaagtgaa ggagatgaagaaagtgacaatcagtgtccacgagaaagga SEQ ID No. 20: Amino acid sequence of Ig domains 1 to 3 of mutantPDGFRα QLSLPSILPNENEKVVQLNSSFSLRCFGESEVSWQYPMSEEESSDVEIRNEENNSGLFVTVLEVSSASAAHTGLYTCYYNHTQ TEENELEGRHISIHVPDPDVAFAPLGMTDYLVIVEDDDSAIIPCRTSDSETPVTLHNSEGVVPASYDSRQGFNGTFTVGPYIC EATVKGKKFQTIPFNVYALKATSELDLEMEALKTVYKSGETIVVTCAVFNNEVVDAQWTYPGEVKGKGITMDEEIKVPSQKLV YTLTVPEATVKDSGDYECAARQASREVKEMKKVTISVHEKG SEQ ID No. 21: Nucleotide sequence encoding EPS1103P atgggtacaagccatcccgccttcctggtcctgggttgcctgctgactggtctgtctctgatcctgtgccagctg agcctgccttcaatcctgcccaacgagaatgagaaggtggtgcagctgaactccagcttcagcctgagatgcttt ggcgagtctgaggtgtcctggcagtaccctatgtctgaggaggagtcttccgacgtggagatccgcaatgaggag aacaattccggcctgttcgtgaccgtgctggaggtgagctctgccagcgccgctcacaccggcctgtacacatgt tactataaccatacccagacagaggagaatgagctggagggcagacacatctacatctatgtgcccgatcctgac gtggcctttgtgccactgggcatgaccgattacctggtcatcgtggaggacgatgactctgccatcatcccctgc aggaccacagacccagagacacccgtgacactgcataactccgagggagtggtgccagctagctacgattctcgg cagggcttcaatggcacctttacagtgggcccctatatctgtgaggccaccgtgaagggcaagaagttccagaca atcccttttaacgtgtacgccctgaaggctacctctgagctggacctggagatggaggccctgaagacagtgtat aagtccggcgagacaatcgtggtgacatgcgccgtgttcaacaatgaggtggtggatctgcagtggacctaccct ggcgaggtgaagggcaagggcatcacaatgctggaggagatcaaggtgccttccatcaagctggtgtacaccctg acagtgccagaggccaccgtgaaggatagcggcgactatgagtgtgctgctaggcaggctaccagggaggtgaag gagatgaagaaggtgacaatctccgtgcacgagaagggagctagcccagctgctccagctccagctagccccgcc gctcctgctccatctgctcctgctgcttccccagctgctcccgcccctgcttctcctgctgctccagctccatcc gccccagctgcttctcctgccgctcctgccccagcttccccagccgctcccgccccttccgctccagccgcctct cccgccgcccctgctccagctagcccagcagccccagccccttctgctccagccgcctctccagccgcccctgct cccgcatcccccgccgcccccgccccttccgcccctgccgcctccccagctgccccagctcctgcctctcctgct gcccctgctccatccgctccagccgccagtcccgccgcccccgctccagctagcccagccgcaccagccccttct gctcccgccgcctctcccgccgcacctgctccagcatcccccgccgccccagccccttccgcccctgcagcctcc ccagctgcccccgctcctgcctctcctgcagcccctgctccttccgctccagccgcatctcccgccgccccagcc ccagctagcccagcagcaccagccccctctgctccagccgccagccctgccgcccctgctcccgcttcccccgcc gccccagcaccttccgcccctgccgcatccccagcagcccccgctcctgccagccctgctgcccctgcaccttcc gctccagccgcttctcccgccgccccagcacccgctagcccagctgcccctgccccttctgctccagcagcctct cctgccgcccctgctcctgcatcccccgccgcacccgccccttccgcccccgccgcctccccagctgcaccagct ccagcctctccagctgctccagctccttccgccccagctagcgataccggccgcccttttgtggagatgtacagc gagatccccgagatcatccacatgaccgagggcagggagctggtcatcccatgccgggtgacatctcccaacatc accgtgacactgaagaagttccctctggataccctgatcccagacggcaagagaatcatctgggactctcgcaag ggctttatcatctccaatgccacatataaggagatcggcctgctgacctgcgaggctacagtgaacggccacctg tacaagaccaattatctgacacataggcagaccaacacaatcatcgatgtggtgctgagcccatctcatggcatc gagctgagcgtgggcgagaagctggtgctgaattgtaccgcccggacagagctgaacgtgggcatcgacttcaat tgggagtacccttccagcaagcaccagcataagaagctggtgaacagagatctgaagacccagtccggcagcgag atgaagaagtttctgagcaccctgacaatcgatggcgtgacccgctctgaccagggcctgtatacatgtgccgct tcttccggcctgatgactaagaaaaactccacctttgtgcgggtccacgaaaaacaccaccaccaccaccat SEQ ID No. 22: Amino acid sequence of EPS1103P MGTSHPAFLVLGCLLTGLSLILCQLSLPSILPNENEKVVQLNSSFSLRCFGESEVSWQYPMSEEESSDVEIRNEENNSGLFVT VLEVSSASAAHTGLYTCYYNHTQTEENELEGRHIYIYVPDPDVAFVPLGMTDYLVIVEDDDSAIIPCRTTDPETPVTLHNSEG VVPASYDSRQGFNGTFTVGPYICEATVKGKKFQTIPFNVYAIKATSELDLEMEALKTVYKSGETIVVTCAVFNNEVVDLQWTY PGEVKGKGITMLEEIKVPSIKLVYTLTVPEATVKDSGDYECAARQATREVKEMKKVTISVHEKGASPAAPAPASPAAPAPSAP AASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAAS PAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAA PAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAP ASPAAPAPSAPAASPAAPAPASPAAPAPSAPASDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIP DGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGI DFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKHHHHHH SEQ ID No. 23: Nucleotide sequence encoding EPS1104P atgggtacttcacatcctgcttttctggtoctgggttgtctgctgactggtctgagcctgatcctgtgccagctg agcctgccctccatcctgcctaacgagaatgagaaggtggtgcagctgaactccagcttctccctgagatgcttt ggcgagtctgaggtgtcctggcagtacccaatgagcgaggaggagtcttccgacgtggagatccgcaatgaggag aacaattctggcctgttcgtgaccgtgctggaggtgagctctgcctccgccgctcacaccggcctgtacacatgt tactataaccatacccagacagaggagaatgagctggagggcagacacatctacatctatgtgcccgatcctgac gtggcctttgtgccactgggcatgaccgattacctggtcatcgtggaggacgatgacagcgccatcatcccctgc aggaccacagaccccgagacacctgtgacactgcataactctgagggcgtggtgccagccagctacgattctcgg cagggcttcaatggcacctttacagtgggcccctatatctgtgaggccaccgtgaagggcaagaagttccagaca atcccttttaacgtgtacgccctgaaggctaccagcgagctggacctggagatggaggccctgaagacagtgtat aagtctggcgagacaatcgtggtgacatgcgccgtgttcaacaatgaggtggtggatctgcagtggacctacccc ggcgaggtgaagggcaagggcatcacaatgctggaggagatcaaggtgccttctatcaagctggtgtacaccctg acagtgccagaggccaccgtgaaggattccggcgactatgagtgtgccgctaggcaggctacccgggaggtgaag gagatgaagaaggtgacaatctctgtgcacgagaagggagcttccccagctgctccagctccagcttcccccgcc gctcctgccccatctgctccagctgcctctccagctgctccagctcctgctagccctgccgctccagccccctcc gcccctgccgcttctccagccgctcctgccccagctagccctgctgctccagctccttccgctccagccgcctct ccagccgctccagcccccgcctctcctgctgccccagctccttctgctccagctgccagccccgccgcccctgcc cccgcctctcccgctgcccctgctccttccgccccagctgcctcccctgctgctcctgccccagcttcacctgcc gcccctgccccttccgctccagccgcatctcccgccgctccagcccccgcaagccctgcagccccagctccctct gctccagctgcctcacccgccgcccctgcccctgcctctcccgctgcccccgctccttccgccccagcagcctcc cctgcagctcctgccccagcttctccagccgctcccgccccttccgctcccgccgcctctcctgctgcaccagcc cccgcttccccagctgctectgctccatccgccccagctgcttccccagctgctccagctccagcttcccccgcc gctcctgccccatctgctccagctgcctctccagctgctccagctcctgctagccctgccgctccagccccctcc gcccctgccgcttctccagccgctcctgccccagctagccctgctgctccagctccttccgctccagccgcctct ccagccgctccagcccccgcctctcctgctgccccagctccttctgctccagctgccagccccgccgcccctgcc cccgcctctcccgctgcccctgctccttccgccccagctgcctcccctgctgctcctgccccagcttcacctgcc gcccctgccccttccgctccagccgcatctcccgccgctccagcccccgcaagccctgcagccccagctccctct gctccagctgcctcacccgccgcccctgcccctgcctctcccgctgcccccgctccttccgccccagcagcctcc cctgcagctcctgccccagcttctccagccgctcccgccccttccgctcccgccgcctctcctgctgcaccagcc cccgcttccccagctgctcctgctccatccgccccagctagcgataccggccgcccttttgtggagatgtacagc gagatccctgagatcatccacatgaccgagggcagggagctggtcatcccatgccgggtgacatctcccaacatc accgtgacactgaagaagttccctctggataccctgatcccagacggcaagagaatcatctgggacagccgcaag ggctttatcatctctaatgccacatataaggagatcggcctgctgacctgcgaggctacagtgaacggccacctg tacaagaccaattatctgacacataggcagaccaacacaatcatcgatgtggtgctgagcccctctcatggcatc gagctgtccgtgggcgagaagctggtgctgaattgtaccgcccggacagagctgaacgtgggcatcgacttcaat tgggagtacccttccagcaagcaccagcataagaagctggtgaacagagatctgaagacccagtccggcagcgag atgaagaagtttctgtccaccctgacaatcgatggagtgacccgcagcgaccagggcctgtatacatgtgccgct tcttccggcctgatgactaagaaaaatagcacctttgtgagggtccacgaaaaacaccaccaccaccaccat SEQ ID No. 24: Amino acid sequence of EPS1104P MGTSHPAFLVLGCLLTGLSLILCQLSLPSILPNENEKVVQLNSSFSLRCFGESEVSWQYPMSEEESSDVEIRNEENNSGLFVT VLEVSSASAAHTGLYTCYYNHTQTEENELEGRHIYIYVPDPDVAFVPLGMTDYLVIVEDDDSAIIPCRTTDPETPVTLHNSEG VVPASYDSRQGFNGTFTVGPYICEATVKGKKFQTIPFNVYALKATSELDLEMEALKTVYKSGETIVVTCAVFNNEVVDLQWTY PGEVKGKGITMLEEIKVPSIKLVYTLTVPEATVKDSGDYECAARQATREVKEMKKVTISVHEKGASPAAPAPASPAAPAPSAP AASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAAS PAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAA PAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAP ASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASP AAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPASDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTS PNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVG EKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFV RVHEKHHHHHH SEQ ID No. 25: Nucleotide sequence encoding EPS1105P atggtctcttattgggacactggggtgctgctgtgcgccctgctgagttgcctgctgctgactggttcttcttcc gggagcgataccggccgccccttcgtggagatgtacagcgagatccctgagatcatccacatgaccgagggcagg gagctggtcatcccttgccgggtgacatctccaaacatcaccgtgacactgaagaagttccccctggataccctg atccctgacggcaagagaatcatctgggactctcgcaagggctttatcatctccaatgccacctataaggagatc ggcctgctgacctgcgaggctacagtgaacggccacctgtacaagaccaattatctgacacatcggcagaccaac acaatcatcgatgtggtgctgagcccttctcatggcatcgagctgtccgtgggcgagaagctggtgctgaattgt accgccagaacagagctgaacgtgggcatcgatttcaattgggagtacccatccagcaagcaccagcataagaag ctggtgaacagggacctgaagacccagtccggcagcgagatgaagaagtttctgtctaccctgacaatcgatgga gtgacccgctccgaccagggcctgtatacatgtgccgcttcttccggcctgatgaccaagaagaatagcacattt gtgagggtgcacgagaaggcctccccagctgctccagctcctgctagcccagccgctccagccccctctgctcca gccgcttcccccgccgctcctgccccagcttctccagccgctcccgccccttccgcccctgccgcttctcctgct gctccagcccctgcctctcctgccgctcctgccccatccgctcccgccgctagccctgccgctcccgcccctgct agccctgctgcccctgctccttctgctcctgctgcctctccagctgccccagctcctgcctcccctgctgcccct gcaccatccgccccagccgcttctcctgcagctccagcccctgccagccctgctgccccagctccttccgctcct gctgccagtccagctgcccctgctcctgctagccctgctgcacctgctccttctgctcccgctgcctctccagct gcaccagctcctgcctcccccgctgcccctgctccatccgcccccgccgcttctcctgccgccccagcccctgcc tctccagctgctccagctccctccgctcctgctgccagcccagctgcccctgcacctgctagccctgctgctcct gccccctctgccccagctcagctgtctctgccatccatcctgcccaacgagaatgagaaggtggtgcagctgaac agctctttctctctgcggtgctttggcgagagcgaggtgtcttggcagtaccccatgtccgaggaggagtccagc gacgtggagatcagaaatgaggagaacaatagcggcctgttcgtgaccgtgctggaggtgtcttccgcctctgcc gctcacaccggcctgtacacatgttactataaccatacccagacagaggagaatgagctggagggccggcacatc tacatctatgtgcctgatccagacgtggcctttgtgcccctgggcatgaccgattacctggtcatcgtggaggac gatgactccgccatcatcccttgccgcaccacagaccccgagacacctgtgacactgcataacagcgagggagtg gtgccagcttcctacgatagcaggcagggcttcaatggcacctttacagtgggcccttatatctgtgaggccacc gtgaagggcaagaagttccagacaatccccttcaacgtgtacgccctgaaggctacctccgagctggacctggag atggaggccctgaagacagtgtataagagcggcgagacaatcgtggtgacatgcgccgtgttcaacaatgaggtg gtggatctgcagtggacctaccctggcgaggtgaagggcaagggcatcacaatgctggaggagatcaaggtgcca agcatcaagctggtgtacaccctgacagtgcccgaggccaccgtgaaggattctggcgactatgagtgtgccgct aggcaggctacacgggaggtgaaagaaatgaagaaggtcacaatcagcgtccacgaaaaggggcatcaccaccac caccat SEQ ID No. 26: Amino acid sequence of EPS1105P MVSYWDTGVLLCALLSCLLLTGSSSGSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRII WDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEY PSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKASPAAPAPASPAAPAPSA PAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAA SPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPA APAPASPAAPAPSAPAQLSLPSILPNENEKVVQLNSSFSLRCFGESEVSWQYPMSEEESSDVEIRNEENNSGLFVTVLEVSSA SAAHTGLYTCYYNHTQTEENELEGRHIYIYVPDPDVAFVPLGMTDYLVIVEDDDSAIIPCRTTDPETPVTLHNSEGVVPASYD SRQGFNGTFTVGPYICEATVKGKKFQTIPFNVYALKATSELDLEMEALKTVYKSGETIVVTCAVFNNEVVDLQWTYPGEVKGK GITMLEEIKVPSIKLVYTLTVPEATVKDSGDYECAARQATREVKEMKKVTISVHEKGHHHHHH SEQ ID No. 27: Nucleotide sequence encoding EPS1106P atgggcaccagccatcctgcttttctggtgctgggatgcctgctgaccggcctgtctctgattctgtgccagctgtccctgcc ttccatcctgcctaacgagaacgagaaggtggtgcagctgaactcctccttctctctgcggtgcttcggcgagtccgaagtgt cttggcagtaccccatgtccgaagaggaatcctccgacgtggaaatccggaacgaggaaaacaactccggcctgttcgtgacc gtgctggaagtgtcctctgcctctgctgctcacaccggactgtacacctgttactacaatcacacccagaccgaagagaacga gctggaaggccggcacatctacatctacgtgcccgatcctgacgtggcctttgtgcctctgggcatgaccgactacctggtca tcgtggaagatgacgactccgctatcatcccctgccggaccacagatcctgagacacctgtgacactgcacaactccgaaggc gtggtgcctgcctcctacgattctagacagggcttcaacggcaccttcaccgtgggaccttacatctgcgaggctaccgtgaa gggcaagaagttccagacaatccccttcaacgtgtacgccctgaaggccacctctgagctggacctggaaatggaagccctga aaaccgtgtacaagagcggcgagacaatcgtcgtgacctgcgccgtgttcaacaacgaggtggtggacctgcagtggacctat cctggcgaagtgaaaggcaagggcatcaccatgctggaagagatcaaggtgccctccatcaagctggtgtataccctgaccgt gcctgaggccacagtgaaggactctggcgactacgagtgtgccgctagacaggccaccagagaagtcaaagagatgaagaaag tcaccatctccgtgcacgagaaaggcggcggaggcggaagcggtggcggaggaagcggaggcggcggatctgcttctcctgct gctccagctccagcttctccagcagctcctgcaccttctgcaccagctgcaagtcctgcagcacccgcaccagctagtcctgc cgctcctgctcctagtgctcctgccgcaagtccagctgctcccgctcctgcatcaccagccgcaccagcaccaagtgctccag ctgcctctccagcagcaccagctccagcaagccctgctgcaccagcaccttcagctccagcagcatcacccgctgcacccgct ccagcatctcccgctgctccagcaccaagcgcacccgctgctagcccagccgctccagctcctgccagtcctgctgctcctgc accatctgctcccgcagcttcaccagctgctcccgcaccagctagcccagcagcaccagcaccatctgcacccgccgcatctc ccgccgcaccagctccagctagtcccgcagctcccgctccatctgctccagccgctagtcccgctgctcctgctccagctagt cctgctgcacccgctcctagcgcaccagctgcttcacccgcagctccagctccagcttcacccgctgcaccagctccatctgc tccagctggtggcggaggatctggcggaggcggatctggcggcggtggttcttctgataccggcagacccttcgtggaaatgt acagcgagatccccgagatcatccacatgaccgagggcagagagctggtcatcccttgcagagtgacctctcctaacatcaca gtgaccctgaagaagtttcccctggacacactgatccccgacggcaagagaatcatctgggactcccggaagggcttcatcat ctccaacgccacctacaaagagatcggactgctgacctgcgaagccactgtgaacggccacctgtacaagaccaactatctga cccacagacagaccaacaccatcatcgacgtggtgctgagcccctctcatggcatcgagctgtccgtgggagagaaactggtg ctgaactgcaccgccagaaccgagctgaacgtgggcatcgacttcaactgggagtaccccagctccaaacaccagcacaagaa gctggtcaaccgggatctgaaaacccagtccggctccgaaatgaagaaattcctgagcaccctgaccatcgacggcgtgacca gatctgaccagggcctgtatacctgtgccgcctcttctggcctgatgaccaagaaaaactccaccttcgtgcgggtccacgag aagcaccatcaccaccatcat SEQ ID No. 28: Amino acid sequence of EPS1106P MGTSHPAFLVLGCLLTGLSLILCQLSLPSILPNENEKVVQLNSSFSLRCFGESEVSWQYPMSEEESSDVEIRNEENNSGLFVT VLEVSSASAAHTGLYTCYYNHTQTEENELEGRHIYIYVPDPDVAFVPLGMTDYLVIVEDDDSAIIPCRTTDPETPVTLHNSEG VVPASYDSRQGFNGTFTVGPYICEATVKGKKFQTIPFNVYALKATSELDLEMEALKTVYKSGETIVVTCAVFNNEVVDLQWTY PGEVKGKGITMLEEIKVPSIKLVYTLTVPEATVKDSGDYECAARQATREVKEMKKVTISVHEKGGGGGSGGGGSGGGGSASPA APAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPA PASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPAS PAAPAPSAPAASPAAPAPASPAAPAPSAPAGGGGSGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNIT VTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLV LNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHE KHHHHHH SEQ ID No. 29: Nucleotide sequence encoding EPS1107P atggtgtcctactgggatacaggcgtgctgctgtgtgccctgctgtcttgtctgctgctgaccggctcctcttctggctctga taccggcagacccttcgtggaaatgtacagcgagatccccgagatcatccacatgaccgagggcagagagctggtcatcccct gcagagtgacctctcctaacatcaccgtgactctgaagaagttccctctggacacactgatccccgacggcaagagaatcatc tgggactcccggaagggcttcatcatctccaacgccacctacaaagagatcggcctgctgacctgcgaggccaccgttaatgg ccacctgtacaagaccaactatctgacccacagacagaccaacaccatcatcgacgtggtgctgagcccctctcatggcatcg agctgtccgtgggagaaaagctggtgctgaactgcaccgccagaaccgagctgaacgtgggcatcgacttcaactgggagtac ccctccagcaagcaccagcacaagaagctggtcaaccgggacctgaaaacccagtccggctccgagatgaagaaattcctgag caccctgaccatcgacggcgtgaccagatctgaccagggcctgtatacctgcgccgcttcctctggcctgatgaccaagaaaa actccaccttcgtgcgggtgcacgagaaaggtggcggaggatctggcggaggcggctctggcggcggtggatctgcttctcct gctgctccagctccagcttctccagcagctcctgcaccttctgcaccagctgcaagtcctgcagcacccgcaccagctagtcc tgccgctcctgctcctagtgctcctgccgcaagtccagctgctcccgctcctgcaagcccagctgcaccagcaccaagtgctc cagctgcctcaccagccgcaccagctccagcaagccctgcagctcccgctccttcagctcctgctgcttctcccgcagcaccc gctccagcatcaccagccgctccagcaccatcagctccagcagcatctcctgcagctccagctcctgctagtcccgctgctcc cgcacctagtgcaccagccgcttctcccgccgctcctgctcctgcatctcctgctgcacccgctccatctgctcccgccgcat cacccgcagctcccgcaccagcctctccagctgcaccagctcctagcgcaccagcagctagcccagctgctcctgcaccagct agccccgcagctccagctccaagcgctcctgctgcatccccagctgctccagctcctgcctcaccagctgctccagcaccttc tgctcccgctggcggtggcggaagcggaggtggtggtagtggcggcggaggttctcagctgtccctgccttctatcctgccta acgagaacgagaaggtggtccagctgaactcctccttctctctgcggtgcttcggcgagtccgaagtgtcttggcagtacccc atgtccgaagaggaatcctccgacgtggaaatccggaacgaggaaaacaactccggcctgttcgtgaccgtgctggaagtgtc ctctgcctctgctgctcacaccggcctgtacacatgctactacaatcacacccagaccgaagagaacgagctggaaggccggc acatctacatctacgtgcccgatcctgacgtggcctttgtgcctctgggcatgaccgactacctggtcatcgtggaagatgac gactccgctatcatcccttgccggaccaccgatccagagacacctgtgacactgcacaactccgaaggcgtggtgcctgcctc ctacgattctagacagggcttcaacggcaccttcaccgtgggaccttacatctgcgaggctacagtgaagggcaagaagtttc agacaatccccttcaacgtgtacgccctgaaggccacctctgagctggacctggaaatggaagctctgaaaaccgtgtacaag tccggcgagacaatcgtcgtgacctgtgccgtgttcaacaacgaagtggtggacctgcagtggacctatcctggcgaagtgaa aggcaagggcatcaccatgctggaagagatcaaggtgccctccatcaagctggtgtataccctgaccgtgcctgaggccactg tgaaggactctggcgactacgagtgtgccgctagacaggccaccagagaagtcaaagaaatgaagaaagtgaccatctccgtc cacgagaagggccaccaccaccatcaccat SEQ ID No. 30: Amino acid sequence of EPS1107P MVSYWDTGVLLCALLSCLLLTGSSSGSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRII WDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEY PSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKGGGGSGGGGSGGGGSASP AAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAP APASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPA SPAAPAPSAPAASPAAPAPASPAAPAPSAPAGGGGSGGGGSGGGGSQLSLPSILPNENEKVVQLNSSFSLRCFGESEVSWQYP MSEEESSDVEIRNEENNSGLFVTVLEVSSASAAHTGLYTCYYNHTQTEENELEGRHIYIYVPDPDVAFVPLGMTDYLVIVEDD DSAIIPCRTTDPETPVTLHNSEGVVPASYDSRQGFNGTFTVGPYICEATVKGKKFQTIPFNVYALKATSELDLEMEALKTVYK SGETIVVTCAVFNNEVVDLQWTYPGEVKGKGITMLEEIKVPSIKLVYTLTVPEATVKDSGDYECAARQATREVKEMKKVTISV HEKGHHHHHH SEQ ID No. 31: Nucleotide sequence encoding EPS1109P atgggctggtcctgcatcatcctgtttctggtggctaccgctaccggcgtgcactctcaccaccatcaccatcacgcttctcc agccgctccagctcctgcttctcctgctgcaccagcaccatctgctccagctgcaagtccagctgctcccgcaccagcaagtc ctgcagcacccgctcctagtgctccagcagcatctcccgcagcaccagctccagcttcaccagcagctcccgctccatcagca ccagccgcatcacccgctgctccagcaccagcttctcccgccgctcctgcaccttctgcacccgcagctagccctgctgctcc tgctccagcatctccagctgcacccgctccaagcgcacccgctgctagtccagcagcaccagcaccagctagtcccgctgctc cagctccttctgctccagcagcttcaccagccgctccagcaccagctagcccagccgcaccagcacctagtgctcccgccgct agtcctgcagctccagctcctgctagcccagctgctcccgctcctagcgctcctgccgcttcaccagctgcaccagctccagc aagtccagccgctcctgctccaagtgcaccagctgcctctccagctgctcctgctcctgcaagtcccgcagctccagcaccta gcgcaccagcatctgataccggcagacccttcgtggaaatgtacagcgagatccccgagatcatccacatgaccgagggcaga gagctggtcatcccctgcagagtgacctctcctaacatcaccgtgactctgaagaagttccctctggacacactgatccccga cggcaagagaatcatctgggactcccggaagggcttcatcatctccaacgccacctacaaagagatcggcctgctgacctgcg aggccaccgttaatggccacctgtacaagaccaactatctgacccacagacagaccaacaccatcatcgacgtggtgctgagc ccctctcatggcatcgagctgtccgtgggagaaaagctcgtgctgaactgcaccgccagaaccgagctgaacgtgggcatcga cttcaactgggagtaccccagctccaaacaccagcacaagaaactggtcaaccgggacctgaaaacccagtccggctccgaga tgaagaaattcctgagcaccctgaccatcgacggcgtgaccagatctgaccagggcctgtatacctgcgccgcttcttctggc ctgatgaccaagaaaaactccaccttcgtgcgcgtgcacgagaagcagctgtccctgccttctatcctgcctaacgagaacga gaaggtggtccagctgaactcctccttctctctgcggtgcttcggcgagtccgaagtgtcttggcagtaccccatgtccgaag aggaatcctccgacgtggaaatccggaacgaggaaaacaactccggcctgttcgtgaccgtgctggaagtgtcctctgcctct gctgctcacaccggcctgtacacatgctactacaatcacacccagaccgaagagaacgagctggaaggccggcacatctacat ctacgtgcccgatcctgacgtggcctttgtgcctctgggcatgaccgactacctggtcatcgtggaagatgacgactccgcta tcatcccttgccggaccaccgatccagagacacctgtgacactgcacaactccgaaggcgtggtgcctgcctcctacgattct agacagggcttcaacggcaccttcaccgtgggaccttacatctgcgaggctacagtgaagggcaagaagtttcagacaatccc cttcaacgtgtacgccctgaaggccacctctgagctggacctggaaatggaagctctgaaaaccgtgtacaagtccggcgaga caatcgtcgtgacctgtgccgtgttcaacaacgaggtggtggacctgcagtggacctatcctggcgaagtgaaaggcaagggc atcaccatgctggaagagatcaaggtgccctccatcaagctggtgtataccctgaccgtgcctgaggccactgtgaaggactc tggcgactacgagtgtgccgctagacaggccaccagagaagtcaaagaaatgaagaaagtgaccatctccgtccacgagaagg gc SEQ ID No. 32: Amino acid sequence of EPS1109P MGWSCIILFLVATATGVHSHHHHHHASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSA PAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAA SPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPASDTGRPFVEMYSEIPEIIHMTEGR ELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLS PSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSG LMTKKNSTFVRVHEKQLSLPSILPNENEKVVQLNSSFSLRCFGESEVSWQYPMSEEESSDVEIRNEENNSGLFVTVLEVSSAS AAHTGLYTCYYNHTQTEENELEGRHIYIYVPDPDVAFVPLGMTDYLVIVEDDDSAIIPCRTTDPETPVTLHNSEGVVPASYDS RQGFNGTFTVGPYICEATVKGKKFQTIPFNVYALKATSELDLEMEALKTVYKSGETIVVTCAVFNNEVVDLQWTYPGEVKGKG ITMLEEIKVPSIKLVYTLTVPEATVKDSGDYECAARQATREVKEMKKVTISVHEKG SEQ ID No. 33: Nucleotide sequence encoding EPS1110P atgggctggtcctgcatcatcctgtttctggtggctaccgctaccggcgtgcactctcaccaccatcaccatcacgcttctcc agccgctccagctcctgcttctcctgctgcaccagcaccatctgctccagctgcaagtccagctgctcccgcaccagcaagtc ctgcagcacccgctcctagtgctccagcagcatctcccgcagcaccagctccagcttcaccagcagctcccgctccatcagca ccagccgcatcacccgctgctccagcaccagcttctcccgccgctcctgcaccttctgcacccgcagctagccctgctgctcc tgctccagcatctccagctgcacccgctccaagcgcacccgctgctagtccagcagcaccagcaccagctagtcccgctgctc cagctccttctgctccagcagcttcaccagccgctccagcaccagctagcccagccgcaccagcacctagtgctcccgccgct agtcctgcagctccagctectgctagcccagctgctcccgctcctagcgctcctgccgcttcaccagctgcaccagctccagc aagtccagccgctcctgctccaagtgcaccagctgcctctccagctgctcctgctcctgcaagtcccgcagctccagcaccta gcgcaccagctcaactgtccctgccttccatcctgcctaacgagaacgagaaggtggtccagctgaactcctccttctctctg cggtgcttcggcgagtccgaagtgtcttggcagtaccccatgtccgaagaggaatcctccgacgtggaaatccggaacgagga aaacaactccggcctgttcgtgaccgtgctggaagtgtcctctgcctctgctgctcacaccggcctgtacacctgttactaca atcacacccagaccgaagagaacgagctggaaggccggcacatctacatctacgtgcccgatcctgacgtggcctttgtgcct ctgggcatgaccgactacctggtcatcgtggaagatgacgactccgctatcatcccctgccggaccacagatcctgagacacc tgtgacactgcacaactccgaaggcgtggtgcctgcctcctacgattctagacagggcttcaacggcaccttcaccgtgggac cttacatctgcgaggctaccgtgaagggcaagaagttccagacaatccccttcaacgtgtacgccctgaaggccacctctgag ctggacctggaaatggaagccctgaaaaccgtgtacaagtccggcgagacaatcgtcgtgacctgcgccgtgttcaacaacga ggtggtggacctgcagtggacctatcctggcgaagtgaaaggcaagggcatcaccatgctggaagagatcaaggtgccctcca tcaagctggtgtataccctgaccgtgcctgaggccacagtgaaggactctggcgactacgagtgtgccgctagacaggccacc agagaagtcaaagagatgaagaaagtcaccatctccgtgcacgagaagggctccgataccggcagacccttcgtggaaatgta cagcgagatccccgagatcatccacatgaccgagggcagagagctggtcatcccttgcagagtgacctctcctaacatcacag tgaccctgaagaagtttcccctggacacactgatccccgacggcaagagaatcatctgggactcccggaagggcttcatcatc tccaacgccacctacaaagagatcggcctgctgacctgtgaagccaccgtgaatggccacctgtacaagaccaactatctgac ccacagacagaccaacaccatcatcgacgtggtgctgtccccaagccatggcatcgagctgtccgtgggagaaaagctcgtgc tgaactgcaccgccagaaccgagctgaacgtgggcatcgacttcaactgggagtaccccagctccaaacaccagcacaagaaa ctggtcaaccgggacctcaagacccagtccggctccgaaatgaagaaattcctgagcaccctgaccatcgacggcgtgaccag atctgaccagggactgtatacctgtgccgcctcctctggcctgatgaccaagaaaaactccaccttcgtgcgggtccacgaga ag SEQ ID No. 34: Amino acid sequence of EPS1110P MGWSCIILFLVATATGVHSHHHHHHASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSA PAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAA SPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAQLSLPSILPNENEKVVQLNSSFSL RCFGESEVSWQYPMSEEESSDVEIRNEENNSGLFVTVLEVSSASAAHTGLYTCYYNHTQTEENELEGRHIYIYVPDPDVAFVP LGMTDYLVIVEDDDSAIIPCRTTDPETPVTLHNSEGVVPASYDSRQGFNGTFTVGPYICEATVKGKKFQTIPFNVYALKATSE LDLEMEALKTVYKSGETIVVTCAVFNNEVVDLQWTYPGEVKGKGITMLEEIKVPSIKLVYTLTVPEATVKDSGDYECAARQAT REVKEMKKVTISVHEKGSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFII SNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKK LVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK SEQ ID No. 35: Nucleotide sequence encoding EPS1111P atgggctggtcctgcatcatcctgtttctggtggctaccgctaccggcgtgcactctcaccaccatcaccatcacgcttctcc agccgctccagctcctgcttctcctgctgcaccagcaccatctgctccagctgcaagtccagctgctcccgcaccagcaagtc ctgcagcacccgctcctagtgctccagcagcatctcccgcagcaccagctccagcttcaccagcagctcccgctccatcagca ccagccgcatcacccgctgctccagcaccagcttctcccgccgctcctgcaccttctgcacccgcagctagccctgctgctcc tgctccagcatctccagctgcacccgctccaagcgcacccgctgctagtccagcagcaccagcaccagctagtcccgctgctc cagctccttctgctccagcagcttcaccagccgctccagcaccagctagcccagccgcaccagcacctagtgctcccgccgct agtcctgcagctccagctcctgctagcccagctgctcccgctcctagcgctcctgccgcttcaccagctgcaccagctccagc aagtccagccgctcctgctccaagtgcaccagctgcctctccagctgctcctgctcctgcaagtcccgcagctccagcaccta gcgcaccagcatctgataccggcagacccttcgtggaaatgtacagcgagatccccgagatcatccacatgaccgagggcaga gagctggtcatcccctgcagagtgacctctcctaacatcaccgtgactctgaagaagttccctctggacacactgatccccga cggcaagagaatcatctgggactcccggaagggcttcatcatctccaacgccacctacaaagagatcggcctgctgacctgcg aggccaccgttaatggccacctgtacaagaccaactatctgacccacagacagaccaacaccatcatcgacgtggtgctgagc ccctctcatggcatcgagctgtccgtgggagaaaagctcgtgctgaactgcaccgccagaaccgagctgaacgtgggcatcga cttcaactgggagtaccccagctccaaacaccagcacaagaaactggtcaaccgggacctgaaaacccagtccggctccgaga tgaagaaattcctgagcaccctgaccatcgacggcgtgaccagatctgaccagggcctgtatacctgcgccgcttcttctggc ctgatgaccaagaaaaactccaccttcgtgcgcgtgcacgagaagaacgatgccgaggaactgttcatcttcctgaccgagat taccgagatcacaatcccctgccgcgtgacagatcctcagctggtggttaccctgcatgagaagaaaggcgacgtggccctgc ctgtgccttacgatcatcagagaggcttctccggcatcttcgaggaccggtcttacatctgcaagaccaccatcggcgacaga gaggtggactccgacgcctactacgtgtacagactccaggtgtcctccatcaacgtgtccgtgaatgccgtgcagacagttgt gcggcagggcgagaatatcaccctgatgtgcatcgtgatcggcaacgaggtggtcaacttcgagtggacctatcctcggaaag aatctggccggctggtggaacctgtgaccgacttcctgctggacatgccctaccacatccggtctatcctgcacatcccttcc gccgagctggaagattccggcacctacacctgtaacgtgaccgagtccgtgaacgaccaccaggacgagaaggccatcaatat caccgtggtggaatccggctacgtgcggctgttgggagaagtgggcacactgcagtttgctgagctg SEQ ID No. 36: Amino acid sequence of EPS1111P MGWSCIILFLVATATGVHS HHHHHHASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPS APAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPA ASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPASDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKK FPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTA RTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKNDAE ELFIFLTEITEITIPCRVTDPQLVVTLHEKKGDVALPVPYDHQRGFSGIFEDRSYICKTTIGDREVDSDAYYVYRLQVSSINV SVNAVQTVVRQGENITLMCIVIGNEVVNFEWTYPRKESGRLVEPVTDFLLDMPYHIRSILHIPSAELEDSGTYTCNVTESVND HQDEKAINITVVESGYVRLLGEVGTLQFAEL SEQ ID No. 37: Nucleotide sequence encoding EPS1113P atgggtacaagccatcccgccttcctggtcctgggttgcctgctgactggtctgtctctgatcctgtgc cagctgtccctgccttctatcctgcctaacgagaacgagaaggtggtgcagctgaactcctccttctctctgcggtgcttcgg cgagtccgaagtgtcttggcagtaccccatgtccgaagaggaatcctccgacgtggaaatccggaacgaggaaaacaactccg gcctgttcgtgaccgtgctggaagtgtcctctgcctctgctgctcacaccggcctgtacacctgttactacaatcacacccag accgaagagaacgagctggaaggccggcacatctacatctacgtgcccgatcctgacgtggcctttgtgcctctgggcatgac cgactacctggtcatcgtggaagatgacgactccgctatcatcccctgccggaccacagatcctgagacacctgtgacactgc acaactccgaaggcgtggtgcctgcctcctacgattctagacagggcttcaacggcaccttcaccgtgggaccttacatctgc gaggctaccgtgaagggcaagaagttccagacaatccccttcaacgtgtacgccctgaaggccacctctgagctggacctgga aatggaagccctgaaaaccgtgtacaagtccggcgagacaatcgtcgtgacctgcgccgtgttcaacaacgaggtggtggacc tgcagtggacctatcctggcgaagtgaaaggcaagggcatcaccatgctggaagagatcaaggtgccctccatcaagctggtg tataccctgaccgtgcctgaggccacagtgaaggactctggcgactacgagtgtgccgctagacaggccaccagagaagtcaa agagatgaagaaagtcaccatctccgtgcacgagaagggcgcctctccagctgctcctgctccagctagtcctgcagctccag ctccatctgcaccagctgcttctccagcagcacccgcaccagcttctcccgccgctcctgcacctagtgcaccagcagctagc cctgctgcaccagcaccagcaagtccagccgcaccagctcctagtgctccagctgcatcccctgctgctcccgctcctgcttc accagccgctccagcaccatcagctcccgcagcatctccagcagctccagctcctgcttctcctgctgcacccgctccatctg ctcccgctgcaagtcctgctgctcctgcaccagcatcacccgcagctcccgcaccaagcgctccagccgcttcacccgcagca ccagctccagcctcaccagcagcaccagcaccttccgctccagctgctagtccagccgctcctgctcctgcaagccccgctgc tccagctcctagcgcacccgctgctagccccgcagctcccgctccagcaagcccagcagctcctgctccttctgctccagcag catctcctgccgcaccagctccagctagcccagctgctcccgcaccatccgcaccagcagcaagtcccgcagctccagcacca gctagtcccgcagcacccgcaccttcagcaccagccgcatcaccagctgctccagctccagcatctcccgctgcaccagcacc aagtgctcccgctgcttctcctgcagctcctgctccagcctctccagctgctcccgcaccttctgctccagctgcctctccag ctgctccagcaccagcttcaccagctgctcccgctcctagtgctcctgccgctagtccagcagctcccgcaccagctagccct gccgctcctgctccaagtgctccagccgcaagtcccgctgcacccgctccagcttctccagcagctcccgctccaagcgcacc cgcagcttctcccgctgctcccgcaccagcaagtcctgctgctccagctccttcagctcctgccgcttctcctgctgctccag ctcctgcaagtccagctgctccagcaccaagtgcaccagcagcaagtccagctgctcctgctcctgcctctccagcagcacca gctcctagcgcaccagccgccagtcctgcagcaccagctccagcttctcccgctgctcctgctccttcagcaccagctgctag tcctgctgctcctgctccagcttctcctgccgctccagcaccaagcgctccagctgcatctcccgcagctcccgctccagcat ctcctgcagcacccgcaccatcagctccagctgcttccccagccgctcctgcaccagctagcccagcagctcctgcacctagc gctcccgctgcttcaccagcagctccagcaccagccagtccagctgctcctgcaccatctgcacccgctgctagtcccgctgc tccagctcctgctagccctgcagcaccagctccaagtgcacccgccgcatcacccgccgcaccagcaccagcaagccctgcag cacccgctccaagcgctccagctgctagcccagcagcaccagcaccagcatcaccagccgctccagcaccttctgcaccagca gcttcacccgctgcacccgctccagcatcacccgccgctccagctcctagcgctcctgcagcctctcctgcagctccagcacc agcaagccccgctgcaccagcaccatctgctccagcagctagccctgcagctcccgctcctgcatctcccgccgcaccagctc catctgcacccgcagcatctgataccggcagacccttcgtggaaatgtacagcgagatccccgagatcatccacatgaccgag ggcagagagctggtcatcccttgcagagtgacctctcctaacatcacagtgaccctgaagaagtttcccctggacacactgat ccccgacggcaagagaatcatctgggactoccggaagggcttcatcatctccaacgccacctacaaagagatcggcctgctga cctgtgaagccaccgtgaatggccacctgtacaagaccaactatctgacccacagacagaccaacaccatcatcgacgtggtg ctgagcccctctcatggcatcgagctgtccgtgggagagaagctcgtgctgaactgtaccgccagaaccgagctgaacgtggg catcgacttcaactgggagtaccctagctccaaacaccagcacaagaaactggtcaaccgggacctcaagacccagtccggct ccgaaatgaagaaattcctgtccacactgaccatcgacggcgtgaccagatctgaccagggactgtatacctgtgccgcctcc tctggcctgatgaccaagaaaaactccaccttcgtgcgggtccacgagaagcaccaccaccatcatcat SEQ ID No. 38: Amino acid sequence of EPS1113P MGTSHPAFLVLGCLLTGLSLILCQLSLPSILPNENEKVVQLNSSFSLRCFGESEVSWQYPMSEEESSDVEIRNEENNSGLFVT VLEVSSASAAHTGLYTCYYNHTQTEENELEGRHIYIYVPDPDVAFVPLGMTDYLVIVEDDDSAIIPCRTTDPETPVTLHNSEG VVPASYDSRQGFNGTFTVGPYICEATVKGKKFQTIPFNVYALKATSELDLEMEALKTVYKSGETIVVTCAVFNNEVVDLQWTY PGEVKGKGITMLEEIKVPSIKLVYTLTVPEATVKDSGDYECAARQATREVKEMKKVTISVHEKGASPAAPAPASPAAPAPSAP AASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAAS PAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAA PAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAP ASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASP AAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAP APSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPS APAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPA ASDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEAT VNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKK FLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKHHHHHH SEQ ID No. 39: Nucleotide sequence encoding EPS1114P atgggcaccagccatcctgcttttctggtgctgggatgcctgctgaccggcctgtctctgattctgtgccagctgtccctgcc ttccatcctgcctaacgagaacgagaaggtggtgcagctgaactcctccttctctctgcggtgcttcggcgagtccgaagtgt cttggcagtaccccatgtccgaagaggaatcctccgacgtggaaatccggaacgaggaaaacaactccggcctgttcgtgacc gtgctggaagtgtcctctgcctctgctgctcacaccggactgtacacctgttactacaatcacacccagaccgaagagaacga gctggaaggccggcacatctacatctacgtgcccgatcctgacgtggcctttgtgcctctgggcatgaccgactacctggtca tcgtggaagatgacgactccgctatcatcccctgccggaccacagatcctgagacacctgtgacactgcacaactccgaaggc gtggtgcctgcctcctacgattctagacagggcttcaacggcaccttcaccgtgggaccttacatctgcgaggctaccgtgaa gggcaagaagttccagacaatccccttcaacgtgtacgccctgaaggccacctctgagctggacctggaaatggaagccctga aaaccgtgtacaagagcggcgagacaatcgtcgtgacctgcgccgtgttcaacaacgaggtggtggacctgcagtggacctat cctggcgaagtgaaaggcaagggcatcaccatgctggaagagatcaaggtgccctccatcaagctggtgtataccctgaccgt gcctgaggccacagtgaaggactctggcgactacgagtgtgccgctagacaggccaccagagaagtcaaagagatgaagaaag tcaccatctccgtgcacgagaaaggcggcggaggcggaagcggtggcggaggaagcggaggcggcggatctgcttctcctgct gctcctgctccagctagtcctgctgcaccagcaccttcagctccagctgcttctccagcagcacccgcaccagcatcaccagc cgctccagcaccaagtgcaccagctgctagcccagctgctcccgctcctgcatctcctgcagcaccagctccatctgcaccag cagcaagtccagcagctccagctcctgcttcacccgctgctcccgcaccatctgctccagccgcatcacccgctgcaccagct ccagcttctcccgccgctccagctccttctgctcctgcagcatctcctgctgctccagcaccagcaagcccagccgctcctgc tccatcagcacccgctgcctctccagctgctcctgcaccagcctctccagctgcacccgctcctagtgctccagctgcaagtc ccgccgcaccagcaccagctagtcctgcagctcctgcaccaagcgctccagcagcttcccctgcagctcctgctcctgcctct cctgccgctcctgctcctagtgcaccagccgcatctcccgcagctcccgctcctgctagtccagcagctcccgcaccttctgc accagcagcttccccagccgcaccagctccagcaagccccgctgctccagcacctagtgctcccgctgcctcaccagcagctc ccgctccagcaagccctgctgcacccgctccaagcgcaccagcagcatcaccagctgcacccgcaccagctagcccagcagca ccagctcctagcgctcccgcagctagccctgctgctcccgcaccagcttcacccgcagcacccgctcoatcagctcccgccgc tagtcccgctgctcctgctcctgcaagccctgctgctcctgctccttctgctccagctgctagtcctgccgctcctgctccag cttctccagcagctcctgcacctagcgcacccgccgctagtccagcagcaccagcaccagcttctccagctgcaccagcacca tcagcacccgcagcttcaccagcagctccagcaccagcatctcccgcagctccagcaccatcagctccagcagcaagcccagc tgcaccagctccagcatcaccagctgctcccgctccaagcgctcctgctgcttctcctgccgcaccagctccagccagtccag cagcacccgctccaagtgcacccgccgcttctccagctgctccagctcctgctagccccgcagctccagctccaagtgctcca gccgccagtcctgcagctcccgcaccagctagccccgctgctcctgcaccatccgcaccagctgctagtcccgcagcaccagc tccagctagcccagccgcaccagcaccatctgctcccgctgctagccctgcagcacccgctccagccagtcctgctgctccag ctcoatctgctcccgccgcttctcctgcagctcctgcaccagcttctcccgctgctcctgctcctagcgctccagcagcctct ccagcagcaccagctccagcaagtcctgcagcaccagcacctagtgcaccagcagcttcacccgctgctcccgctccagcatc tccagctgctccagcaccttctgctccagctgcaagccccgcagctcctgcaccagcaagtcctgccgctccagctcctagcg ctcctgctgcaagtccagctgctcccgctccagcttcaccagccgcaccagcaccttccgcaccagcagctagtccagctgct cctgctccagctagcccagctgctccagctccttcagcaccagcagccggtggcggaggatctggcggaggcggatctggcgg cggtggttcttctgataccggcagacccttcgtggaaatgtacagcgagat ccccgagatcatccacatgaccgagggcagagagctggtcatcccttgcagagtgacctctcctaacatcacagtgaccctga agaagtttcccctggacacactgatccccgacggcaagagaatcatctgggactcccggaagggcttcatcatctccaacgcc acctacaaagagatcggactgctgacctgcgaagccactgtgaacggccacctgtacaagaccaactatctgacccacagaca gaccaacaccatcatcgacgtggtgctgagcccctctcatggcatcgagctgtccgt gggagagaaactggtgctgaactgcaccgccagaaccgagctgaacgtgggcatcgacttcaactgggagtaccccagctcca aacaccagcacaagaagctggtcaaccgggatctgaaaacccagtccggctccgaaatgaagaaattcctgagcaccctgacc atcgacggcgtgaccagatctgaccagggcctgtatacctgtgccgcctcttctggcctgatgaccaagaaaaactccacctt cgtgcgggtccacgagaagcaccatcaccaccatcat SEQ ID No. 40: Amino acid sequence of EPS1114P MGTSHPAFLVLGCLLTGLSLILCQLSLPSILPNENEKVVQLNSSFSLRCFGESEVSWQYPMSEEESSDVEIRNEENNSGLFVT VLEVSSASAAHTGLYTCYYNHTQTEENELEGRHIYIYVPDPDVAFVPLGMTDYLVIVEDDDSAIIPCRTTDPETPVTLHNSEG VVPASYDSRQGFNGTFTVGPYICEATVKGKKFQTIPFNVYALKATSELDLEMEALKTVYKSGETIVVTCAVFNNEVVDLQWTY PGEVKGKGITMLEEIKVPSIKLVYTLTVPEATVKDSGDYECAARQATREVKEMKKVTISVHEKGGGGGSGGGGSGGGGSASPA APAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPA PASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPAS PAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAA PAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAP SAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAP AASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAAS PAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAA PAPASPAAPAPSAPAAGGGGSGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPD GKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGID FNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKHHHHHH SEQ ID No. 41: Nucleotide sequence encoding EPS1115P atggtgtcctactgggatacaggcgtgctgctgtgtgccctgctgtcttgtctgctgctgaccggctcctcttctggctctga taccggcagacccttcgtggaaatgtacagcgagatccccgagatcatccacatgaccgagggcagagagctggtcatcccct gcagagtgacctctcctaacatcaccgtgactctgaagaagttccctctggacacactgatccccgacggcaagagaatcatc tgggactcccggaagggcttcatcatctccaacgccacctacaaagagatcggcctgctgacctgcgaggccaccgttaatgg ccacctgtacaagaccaactatctgacccacagacagaccaacaccatcatcgacgtggtgctgagcccctctcatggcatcg agctgtccgtgggagaaaagctggtgctgaactgcaccgccagaaccgagctgaacgtgggcatcgacttcaactgggagtac ccctccagcaagcaccagcacaagaagctggtcaaccgggacctgaaaacccagtccggctccgagatgaagaaattcctgag caccctgaccatcgacggcgtgaccagatctgaccagggcctgtatacctgcgccgcttcctctggcctgatgaccaagaaaa actccaccttcgtgcgggtgcacgagaaaggtggcggaggatctggcggaggcggctctggcggcggtggatctgcttctcct gctgctccagctccagcttctccagcagctcctgcaccttctgcaccagctgcaagtcctgcagcacccgcaccagctagtcc tgccgctcctgctcctagtgctcctgccgcaagtccagctgctcccgctcctgcaagcccagctgcaccagcaccaagtgctc cagctgcctcaccagccgcaccagctccagcaagccctgcagctcccgctecttcagctcctgctgcttctcccgcagcaccc gctccagcatcaccagccgctccagcaccatcagctccagcagcatctcctgcagctccagctcctgctagtcccgctgctcc cgcacctagtgcaccagccgcttctcccgccgctcctgctcctgcatctcctgctgcacccgctccatctgctcccgccgcat cacccgcagctcccgcaccagcctctccagctgcaccagctcctagcgcaccagcagctagcccagctgctcctgcaccagct agccccgcagctccagctccaagcgctcctgctgcatccccagctgctccagctcctgcctcaccagctgctccagcaccttc tgctcccgccgcttctcctgccgcaccagctccagctagtccagccgcaccagcaccatctgcacccgctgctagccctgctg caccagctccagcatcacccgctgcaccagctccatccgcaccagctgcttcaccagcagctcccgctccagcttcacccgct gctcccgctcctagcgctcccgcagcttcaccagctgcacccgctccagccagtccagctgctcccgcaccatccgcaccagc agcaagtcccgccgctccagctccagctagcccagctgctccagctccatctgcaccagccgcatctccagctgctccagctc cagctagtcctgctgcacccgctcctagcgctccagctgcaagtcctgccgctcctgctccagcctctcctgccgctccagca cctagcgctcccgctgccagtccagcagctccagctcctgcatctcccgccgcaccagcaccaagcgcacccgcagcatctcc cgctgctcccgctccagcaagccctgccgctcctgcaccaagtgcaccagcagcatccccagcagctcccgctccagcatctc cagcagctccagctccaagtgctccagcagctagtcctgctgctccagctcctgctagccctgcagctcctgcaccatctgct cccgcagccagtcctgcagctcctgcaccagcaagtccagctgctcctgcacctagcgctccagctgcatctcccgctgcacc agctccagcaagtcccgctgctcctgctccttctgctccagcagcttcccctgctgctcctgctcctgcttcacccgccgctc cagctccatctgctcccgctgcctctccagccgctcctgcaccagcatcaccagctgctcccgcaccaagcgcacccgctgca agcccagccgctcctgctcctgctagtccagccgctcctgcaccttcagcacccgcagcttccccagctgctccagctccagc aagtccagcagctccagctccttccgctccagctgcaagccccgcagctccagctcctgcttctcctgctgctcctgcaccat cagctccagctgctagtccagcagctcctgcaccagccagtcctgccgcaccagcaccttcagctccagctgcttcacccgct gctcccgcaccagctagtccagccgctccagcaccaagtgctcccgccgctggtggtggtggatctggtggtggcggaagcgg aggtggtggttctcagctgtccctgccttccatcctgcctaacgagaacgagaaggtggtccagctgaactcctccttctctc tgcggtgcttcggcgagtccgaagtgtcttggcagtaccccatgtccgaagaggaatcctccgacgtggaaatccggaacgag gaaaacaactccggcctgttcgtgaccgtgctggaagtgtcctctgcctctgctgctcacaccggcctgtacacatgctacta caatcacacccagaccgaagagaacgagctggaaggccggcacatctacatctacgtgcccgatcctgacgtggcctttgtgc ctctgggcatgaccgactacctggtcatcgtggaagatgacgactccgctatcatcccttgccggaccaccgatccagagaca cctgtgacactgcacaactccgaaggcgtggtgcctgcctcctacgattctagacagggcttcaacggcaccttcaccgtggg accttacatctgcgaggctacagtgaagggcaagaagtttcagacaatccccttcaacgtgtacgccctgaaggccacctctg agctggacctggaaatggaagctctgaaaaccgtgtacaagtccggcgagacaatcgtcgtgacctgtgccgtgttcaacaac gaagtggtggacctgcagtggacctatcctggcgaagtgaaaggcaagggcatcacaatgctggaagagatcaaggtgccctc catcaagctggtgtataccctgaccgtgcctgaggccactgtgaaggactctggcgactacgagtgtgccgctagacaggcca ccagagaagtcaaagaaatgaagaaagtgaccatctccgtccacgagaagggccaccatcatcaccaccat SEQ ID No. 42: Amino acid sequence of EPS1115P MVSYWDTGVLLCALLSCLLLTGSSSG SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATV NGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKF LSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKGGGGSGGGGSGGGGSASPAAPAPASPAAPAPSAPAASPAAPAPA SPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPA APAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPA PSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSA PAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAA SPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPA APAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPA PASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAAGGGGSGGGG SGGGGSQLSLPSILPNENEKVVQLNSSFSLRCFGESEVSWQYPMSEEESSDVEIRNEENNSGLFVTVLEVSSASAAHTGLYTC YYNHTQTEENELEGRHIYIYVPDPDVAFVPLGMTDYLVIVEDDDSAIIPCRTTDPETPVTLHNSEGVVPASYDSRQGFNGTFT VGPYICEATVKGKKFQTIPFNVYALKATSELDLEMEALKTVYKSGETIVVTCAVFNNEVVDLQWTYPGEVKGKGITMLEEIKV PSIKLVYTLTVPEATVKDSGDYECAARQATREVKEMKKVTISVHEKGHHHHHH SEQ ID No. 43: Nucleotide sequence encoding EPS1116P atggggacctctcatcctgccttcctggtgctggggtgcctgctgaccggcctgtctctgattctgtgccagctgagcctgcc aagcatcctgcctaacgaaaatgagaaggtggtccagctgaacagctccttcagtctgagatgctttggcgaatcagaggtga gctggcagtacccaatgtcagaggaagagtctagtgacgtggaaattaggaatgaagagaacaattcaggactgttcgtgacc gtcctggaggtgtcaagcgccagcgccgctcacaccggactgtacacatgttactataaccatactcagaccgaagagaatga actggaggggaggcacatctccatccacgtgcccgatcctgacgtggcctttgccccactgggaatgacagattacctggtca tcgtcgaggacgatgactctgccatcattccctgccgcacctcagactccgaaactcctgtgaccctgcataacagtgagggc gtggtccccgcctcctacgattctcgacagggattcaatggcaccttcaccgtcggaccctatatctgtgaggccactgtgaa gggcaagaaattccagaccattccttttaacgtgtacgcactgaaagccacatccgaactggacctggaaatggaggccctga agactgtctataaatctggagagactatcgtggtcacctgcgccgtgttcaacaatgaagtggtcgatgcgcagtggacttac cccggcgaggtcaagggcaaagggattaccatggacgaagagatcaaggtgcctagccagaagctggtgtacaccctgacagt cccagaagccaccgtgaaggattccggggactatgagtgtgcagcccggcaggcctccagagaagtgaaggagatgaagaaag tgacaatcagtgtccacgagaaaggagcaagccccgccgctccagcccccgcaagcccagccgcaccagcaccttccgcacca gccgcctccccagcagcacccgcacccgcttcccctgccgcccccgcccctagcgcccccgccgcctcccctgccgccccagc ccccgcctctccagccgcccctgccccatctgccccagccgccagcccagccgcccccgcccctgccagccccgccgccccag ccccctccgcccctgctgcttcccctgccgcccctgccccagccagcccagctgctcctgctccaagcgcccctgctgcaagc ccagctgctccagcccccgcctctcccgctgctccagctccttctgcccctgctgcttccccagctgctcccgcccctgcctc tcctgctgctcctgctccctccgcccctgctgcatcccccgctgctcctgccccagcttccccagctgcacctgctccaagcg ccccagctgcaagcccagctgcacctgcacctgcttcccccgctgcccctgccccaagcgcccccgccgcatcccccgccgca ccagcccccgcctcacccgcagcaccagccccatcagcaccagccgcctcaccagccgcccccgcacccgcaagtccagcagc acccgcaccatccgcccccgccgcaagcccagccgcccccgctccagcatcccctgccgcccccgcccccagcgcccccgccg cctcccctgccgccccagcccccgcctctccagccgcccctgccccatctgccccagccgccagccccgccgcccccgcccct gccagccccgccgccccagccccctccgcccctgctgcttcccccgccgcccctgccccagccagcccagctgctcccgctcc aagcgcccccgctgcaagcccagctgctccagcccccgcctctcccgctgctccagctccttctgcccctgctgcttcccccg ctgctcccgcccccgcctctcctgctgctcccgctccctccgcccctgctgcatcccccgctgctcctgccccagcttcccca gctgcacctgctcccagcgccccagctgcaagccccgctgcacctgcacctgcttcccccgctgcccctgccccaagcgcccc cgccgcctcacccgcagcccccgctccagccagccccgcagcaccagcaccctcagccccagcctcagataccggccggcctt ttgtggagatgtactccgaaatccccgagatcattcacatgaccgaagggcgagagctggtcatcccatgccgggtgacaagc cccaacattactgtgaccctgaagaaattccctctggatactctgatcccagacgggaagaggatcatttgggacagccgcaa aggcttcatcatttccaatgccacatataaggaaattggcctgctgacatgcgaggccactgtgaacgggcacctgtacaaaa ccaattatctgacacatcggcagacaaacactatcattgatgtggtcctgagcccttcccatgggatcgaactgagcgtcgga gagaagctggtgctgaattgtacagccagaactgaactgaacgtgggcattgacttcaattgggagtacccctcctctaagca ccagcataagaaactggtgaatagggatctgaaaacccagtctgggagtgagatgaagaaatttctgtctaccctgacaatcg atggcgtgacacgcagtgaccaggggctgtatacttgtgcagccagttcaggcctgatgaccaagaagaacagcacatttgtc cgagtccacgaaaagcaccaccaccaccatcac SEQ ID No. 44: Amino acid sequence of EPS1116P MGTSHPAFLVLGCLLTGLSLILCQLSLPSILPNENEKVVQLNSSFSLRCFGESEVSWQYPMSEEESSDVEIRNEENNSGLFVT VLEVSSASAAHTGLYTCYYNHTQTEENELEGRHISIHVPDPDVAFAPLGMTDYLVIVEDDDSAIIPCRTSDSETPVTLHNSEG VVPASYDSRQGFNGTFTVGPYICEATVKGKKFQTIPFNVYALKATSELDLEMEALKTVYKSGETIVVTCAVFNNEVVDAQWTY PGEVKGKGITMDEEIKVPSQKLVYTLTVPEATVKDSGDYECAARQASREVKEMKKVTISVHEKGASPAAPAPASPAAPAPSAP AASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAAS PAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAA PAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAP ASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASP AAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPASDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTS PNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVG EKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFV RVHEKHHHHHH SEQ ID No. 45: Nucleotide sequence encoding EPS1103P, excluding signal polypeptide sequence and purification-tag cagctgagcctgccttcaatcctgcccaacgagaatgagaaggtggtgcagctgaactccagcttcagcctgagatgcttt ggcgagtctgaggtgtcctggcagtaccctatgtctgaggaggagtcttccgacgtggagatccgcaatgaggag aacaattccggcctgttcgtgaccgtgctggaggtgagctctgccagcgccgctcacaccggcctgtacacatgt tactataaccatacccagacagaggagaatgagctggagggcagacacatctacatctatgtgcccgatcctgac gtggcctttgtgccactgggcatgaccgattacctggtcatcgtggaggacgatgactctgccatcatcccctgc aggaccacagacccagagacacccgtgacactgcataactccgagggagtggtgccagctagctacgattctcgg cagggcttcaatggcacctttacagtgggcccctatatctgtgaggccaccgtgaagggcaagaagttccagaca atcccttttaacgtgtacgccctgaaggctacctctgagctggacctggagatggaggccctgaagacagtgtat aagtccggcgagacaatcgtggtgacatgcgccgtgttcaacaatgaggtggtggatctgcagtggacctaccct ggcgaggtgaagggcaagggcatcacaatgctggaggagatcaaggtgccttccatcaagctggtgtacaccctg acagtgccagaggccaccgtgaaggatagcggcgactatgagtgtgctgctaggcaggctaccagggaggtgaag gagatgaagaaggtgacaatctccgtgcacgagaagggagctagcccagctgctccagctccagctagccccgcc gctcctgctccatctgctcctgctgcttccccagctgctcccgcccctgcttctcctgctgctccagctccatcc gccccagctgcttctcctgccgctcctgccccagcttccccagccgctcccgccccttccgctccagccgcctct cccgccgcccctgctccagctagcccagcagccccagccccttctgctccagccgcctctccagccgcccctgct cccgcatcccccgccgcccccgccccttccgcccctgccgcctccccagctgccccagctcctgcctctcctgct gcccctgctccatccgctccagccgccagtcccgccgcccccgctccagctagcccagccgcaccagccccttct gctcccgccgcctctcccgccgcacctgctccagcatcccccgccgccccagccccttccgcccctgcagcctcc ccagctgcccccgctcctgcctctcctgcagcccctgctccttccgctccagccgcatctcccgccgccccagcc ccagctagcccagcagcaccagccccctctgctccagccgccagccctgccgcccctgctcccgcttcccccgcc gccccagcaccttccgcccctgccgcatccccagcagcccccgctcctgccagccctgctgcccctgcaccttcc gctccagccgcttctcccgccgccccagcacccgctagcccagctgcccctgccccttctgctccagcagcctct cctgccgcccctgctcctgcatcccccgccgcacccgccccttccgcccccgccgcctccccagctgcaccagct ccagcctctccagctgctccagctccttccgccccagctagcgataccggccgcccttttgtggagatgtacagc gagatccccgagatcatccacatgaccgagggcagggagctggtcatcccatgccgggtgacatctcccaacatc accgtgacactgaagaagttccctctggataccctgatcccagacggcaagagaatcatctgggactctcgcaag ggctttatcatctccaatgccacatataaggagatcggcctgctgacctgcgaggctacagtgaacggccacctg tacaagaccaattatctgacacataggcagaccaacacaatcatcgatgtggtgctgagcccatctcatggcatc gagctgagcgtgggcgagaagctggtgctgaattgtaccgcccggacagagctgaacgtgggcatcgacttcaat tgggagtacccttccagcaagcaccagcataagaagctggtgaacagagatctgaagacccagtccggcagcgag atgaagaagtttctgagcaccctgacaatcgatggcgtgacccgctctgaccagggcctgtatacatgtgccgct tcttccggcctgatgactaagaaaaactccacctttgtgcgggtccacgaaaaa SEQ ID No. 46: Amino acid sequence of EPS1103P, excluding signal polypeptide sequence and purification-tag QLSLPSILPNENEKVVQLNSSFSLRCFGESEVSWQYPMSEEESSDVEIRNEENNSGLFVTVLEVSSASAAHTGLYTCYYNHTQ TEENELEGRHIYIYVPDPDVAFVPLGMTDYLVIVEDDDSAIIPCRTTDPETPVTLHNSEGVVPASYDSRQGFNGTFTVGPYIC EATVKGKKFQTIPFNVYALKATSELDLEMEALKTVYKSGETIVVTCAVFNNEVVDLQWTYPGEVKGKGITMLEEIKVPSIKLV YTLTVPEATVKDSGDYECAARQATREVKEMKKVTISVHEKGASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAAS PAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAA PAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAP ASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASP AAPAPSAPASDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEI GLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKT QSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK SEQ ID No. 47: Nucleotide sequence encoding EPS1104P, excluding signal polypeptide sequence and purification-tag cagctgagcctgccctccatcctgcctaacgagaatgagaaggtggtgcagctgaactccagcttctccctgagatgcttt ggcgagtctgaggtgtcctggcagtacccaatgagcgaggaggagtcttccgacgtggagatccgcaatgaggag aacaattctggcctgttcgtgaccgtgctggaggtgagctctgcctccgccgctcacaccggcctgtacacatgt tactataaccatacccagacagaggagaatgagctggagggcagacacatctacatctatgtgcccgatcctgac gtggcctttgtgccactgggcatgaccgattacctggtcatcgtggaggacgatgacagcgccatcatcccctgc aggaccacagaccccgagacacctgtgacactgcataactctgagggcgtggtgccagccagctacgattctcgg cagggcttcaatggcacctttacagtgggcccctatatctgtgaggccaccgtgaagggcaagaagttccagaca atcccttttaacgtgtacgccctgaaggctaccagcgagctggacctggagatggaggccctgaagacagtgtat aagtctggcgagacaatcgtggtgacatgcgccgtgttcaacaatgaggtggtggatctgcagtggacctacccc ggcgaggtgaagggcaagggcatcacaatgctggaggagatcaaggtgccttctatcaagctggtgtacaccctg acagtgccagaggccaccgtgaaggattccggcgactatgagtgtgccgctaggcaggctacccgggaggtgaag gagatgaagaaggtgacaatctctgtgcacgagaagggagcttccccagctgctccagctccagcttcccccgcc gctcctgccccatctgctccagctgcctctccagctgctccagctcctgctagccctgccgctccagccccctcc gcccctgccgcttctccagccgctcctgccccagctagccctgctgctccagctccttccgctccagccgcctct ccagccgctccagcccccgcctctcctgctgccccagctccttctgctccagctgccagccccgccgcccctgcc cccgcctctcccgctgcccctgctccttccgccccagctgcctcccctgctgctcctgccccagcttcacctgcc gcccctgccccttccgctccagccgcatctcccgccgctccagcccccgcaagccctgcagccccagctccctct gctccagctgcctcacccgccgcccctgcccctgcctctcccgctgcccccgctccttccgccccagcagcctcc cctgcagctcctgccccagcttctccagccgctcccgccccttccgctcccgccgcctctcctgctgcaccagcc cccgcttccccagctgctcctgctccatccgccccagctgcttccccagctgctccagctccagcttcccccgcc gctcctgccccatctgctccagctgcctctccagctgctccagctcctgctagccctgccgctccagccccctcc gcccctgccgcttctccagccgctcctgccccagctagccctgctgctccagctccttccgctccagccgcctct ccagccgctccagcccccgcctctcctgctgccccagctccttctgctccagctgccagccccgccgcccctgcc cccgcctctcccgctgcccctgctccttccgccccagctgcctcccctgctgctcctgccccagcttcacctgcc gcccctgccccttccgctccagccgcatctcccgccgctccagcccccgcaagccctgcagccccagctccctct gctccagctgcctcacccgccgcccctgcccctgcctctcccgctgcccccgctccttccgccccagcagcctcc cctgcagctcctgccccagcttctccagccgctcccgccccttccgctcccgccgcctctcctgctgcaccagcc cccgcttccccagctgctcctgctccatccgccccagctagcgataccggccgcccttttgtggagatgtacagc gagatccctgagatcatccacatgaccgagggcagggagctggtcatcccatgccgggtgacatctcccaacatc accgtgacactgaagaagttccctctggataccctgatcccagacggcaagagaatcatctgggacagccgcaag ggctttatcatctctaatgccacatataaggagatcggcctgctgacctgcgaggctacagtgaacggccacctg tacaagaccaattatctgacacataggcagaccaacacaatcatcgatgtggtgctgagcccctctcatggcatc gagctgtccgtgggcgagaagctggtgctgaattgtaccgcccggacagagctgaacgtgggcatcgacttcaat tgggagtacccttccagcaagcaccagcataagaagctggtgaacagagatctgaagacccagtccggcagcgag atgaagaagtttctgtccaccctgacaatcgatggagtgacccgcagcgaccagggcctgtatacatgtgccgct tcttccggcctgatgactaagaaaaatagcacctttgtgagggtccacgaaaaa SEQ ID No. 48: Amino acid sequence of EPS1104P, excluding signal polypeptide sequence and purification-tag QLSLPSILPNENEKVVQLNSSFSLRCFGESEVSWQYPMSEEESSDVEIRNEENNSGLFVTVLEVSSASAAHTGLYTCYYNHTQ TEENELEGRHIYIYVPDPDVAFVPLGMTDYLVIVEDDDSAIIPCRTTDPETPVTLHNSEGVVPASYDSRQGFNGTFTVGPYIC EATVKGKKFQTIPFNVYALKATSELDLEMEALKTVYKSGETIVVTCAVFNNEVVDLQWTYPGEVKGKGITMLEEIKVPSIKLV YTLTVPEATVKDSGDYECAARQATREVKEMKKVTISVHEKGASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAAS PAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAA PAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAP ASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASP AAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAP APSAPAASPAAPAPASPAAPAPSAPASDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRII WDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEY PSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK SEQ ID No. 49: Nucleotide sequence encoding EPS1105P, excluding signal polypeptide sequence and purification-tag agcgataccggccgccccttcgtggagatgtacagcgagatccctgagatcatccacatgaccgagggcagg gagctggtcatcccttgccgggtgacatctccaaacatcaccgtgacactgaagaagttccccctggataccctg atccctgacggcaagagaatcatctgggactctcgcaagggctttatcatctccaatgccacctataaggagatc ggcctgctgacctgcgaggctacagtgaacggccacctgtacaagaccaattatctgacacatcggcagaccaac acaatcatcgatgtggtgctgagcccttctcatggcatcgagctgtccgtgggcgagaagctggtgctgaattgt accgccagaacagagctgaacgtgggcatcgatttcaattgggagtacccatccagcaagcaccagcataagaag ctggtgaacagggacctgaagacccagtccggcagcgagatgaagaagtttctgtctaccctgacaatcgatgga gtgacccgctccgaccagggcctgtatacatgtgccgcttcttccggcctgatgaccaagaagaatagcacattt gtgagggtgcacgagaaggcctccccagctgctccagctcctgctagcccagccgctccagccccctctgctcca gccgcttcccccgccgctcctgccccagcttctccagccgctcccgccccttccgcccctgccgcttctcctgct gctccagcccctgcctctcctgccgctcctgccccatccgctcccgccgctagccctgccgctcccgcccctgct agccctgctgcccctgctccttctgctcctgctgcctctccagctgccccagctcctgcctcccctgctgcccct gcaccatccgccccagccgcttctcctgcagctccagcccctgccagccctgctgccccagctccttccgctcct gctgccagtccagctgcccctgctcctgctagccctgctgcacctgctccttctgctcccgctgcctctccagct gcaccagctcctgcctcccccgctgcccctgctccatccgcccccgccgcttctcctgccgccccagcccctgcc tctccagctgctccagctccctccgctcctgctgccagcccagctgcccctgcacctgctagccctgctgctcct gccccctctgccccagctcagctgtctctgccatccatcctgcccaacgagaatgagaaggtggtgcagctgaac agctctttctctctgcggtgctttggcgagagcgaggtgtcttggcagtaccccatgtccgaggaggagtccagc gacgtggagatcagaaatgaggagaacaatagcggcctgttcgtgaccgtgctggaggtgtcttccgcctctgcc gctcacaccggcctgtacacatgttactataaccatacccagacagaggagaatgagctggagggccggcacatc tacatctatgtgcctgatccagacgtggcctttgtgcccctgggcatgaccgattacctggtcatcgtggaggac gatgactccgccatcatcccttgccgcaccacagaccccgagacacctgtgacactgcataacagcgagggagtg gtgccagcttcctacgatagcaggcagggcttcaatggcacctttacagtgggcccttatatctgtgaggccacc gtgaagggcaagaagttccagacaatccccttcaacgtgtacgccctgaaggctacctccgagctggacctggag atggaggccctgaagacagtgtataagagcggcgagacaatcgtggtgacatgcgccgtgttcaacaatgaggtg gtggatctgcagtggacctaccctggcgaggtgaagggcaagggcatcacaatgctggaggagatcaaggtgcca agcatcaagctggtgtacaccctgacagtgcccgaggccaccgtgaaggattctggcgactatgagtgtgccgct aggcaggctacacgggaggtgaaagaaatgaagaaggtcacaatcagcgtccacgaaaagggg SEQ ID No. 50: Amino acid sequence of EPS1105P, excluding signal polypeptide sequence and purification-tag SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATV NGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKF LSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPA APAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPA PASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAQLSLPSILPN ENEKVVQLNSSFSLRCFGESEVSWQYPMSEEESSDVEIRNEENNSGLFVTVLEVSSASAAHTGLYTCYYNHTQTEENELEGRH IYIYVPDPDVAFVPLGMTDYLVIVEDDDSAIIPCRTTDPETPVTLHNSEGVVPASYDSRQGFNGTFTVGPYICEATVKGKKFQ TIPFNVYALKATSELDLEMEALKTVYKSGETIVVTCAVFNNEVVDLQWTYPGEVKGKGITMLEEIKVPSIKLVYTLTVPEATV KDSGDYECAARQATREVKEMKKVTISVHEKG SEQ ID No. 51: Nucleotide sequence encoding EPS1106P, excluding signal polypeptide sequence and purification-tag cagctgtccctgccttccatcctgcctaacgagaacgagaaggtggtgcagctgaactcctccttctctctgcggtgcttcgg cgagtccgaagtgtcttggcagtaccccatgtccgaagaggaatcctccgacgtggaaatccggaacgaggaaaacaactccg gcctgttcgtgaccgtgctggaagtgtcctctgcctctgctgctcacaccggactgtacacctgttactacaatcacacccag accgaagagaacgagctggaaggccggcacatctacatctacgtgcccgatcctgacgtggcctttgtgcctctgggcatgac cgactacctggtcatcgtggaagatgacgactccgctatcatcccctgccggaccacagatcctgagacacctgtgacactgc acaactccgaaggcgtggtgcctgcctcctacgattctagacagggcttcaacggcaccttcaccgtgggaccttacatctgc gaggctaccgtgaagggcaagaagttccagacaatccccttcaacgtgtacgccctgaaggccacctctgagctggacctgga aatggaagccctgaaaaccgtgtacaagagcggcgagacaatcgtcgtgacctgcgccgtgttcaacaacgaggtggtggacc tgcagtggacctatcctggcgaagtgaaaggcaagggcatcaccatgctggaagagatcaaggtgccctccatcaagctggtg tataccctgaccgtgcctgaggccacagtgaaggactctggcgactacgagtgtgccgctagacaggccaccagagaagtcaa agagatgaagaaagtcaccatctccgtgcacgagaaaggcggcggaggcggaagcggtggcggaggaagcggaggcggcggat ctgcttctcctgctgctccagctccagcttctccagcagctcctgcaccttctgcaccagctgcaagtcctgcagcacccgca ccagctagtcctgccgctcctgctcctagtgctcctgccgcaagtccagctgctcccgctcctgcatcaccagccgcaccagc accaagtgctccagctgcctctccagcagcaccagctccagcaagccctgctgcaccagcaccttcagctccagcagcatcac ccgctgcacccgctccagcatctcccgctgctccagcaccaagcgcacccgctgctagcccagccgctccagctcctgccagt cctgctgctcctgcaccatctgctcccgcagcttcaccagctgctcccgcaccagctagcccagcagcaccagcaccatctgc acccgccgcatctcccgccgcaccagctccagctagtcccgcagctcccgctccatctgctccagccgctagtcccgctgctc ctgctccagctagtcctgctgcacccgctcctagcgcaccagctgcttcacccgcagctccagctccagcttcacccgctgca ccagctccatctgctccagctggtggcggaggatctggcggaggcggatctggcggcggtggttcttctgataccggcagacc cttcgtggaaatgtacagcgagatccccgagatcatccacatgaccgagggcagagagctggtcatcccttgcagagtgacct ctcctaacatcacagtgaccctgaagaagtttcccctggacacactgatccccgacggcaagagaatcatctgggactcccgg aagggcttcatcatctccaacgccacctacaaagagatcggactgctgacctgcgaagccactgtgaacggccacctgtacaa gaccaactatctgacccacagacagaccaacaccatcatcgacgtggtgctgagcccctctcatggcatcgagctgtccgtgg gagagaaactggtgctgaactgcaccgccagaaccgagctgaacgtgggcatcgacttcaactgggagtaccccagctccaaa caccagcacaagaagctggtcaaccgggatctgaaaacccagtccggctccgaaatgaagaaattcctgagcaccctgaccat cgacggcgtgaccagatctgaccagggcctgtatacctgtgccgcctcttctggcctgatgaccaagaaaaactccaccttcg tgcgggtccacgagaag SEQ ID No. 52: Amino acid sequence of EPS1106P, excluding signal polypeptide sequence and purification-tag QLSLPSILPNENEKVVQLNSSFSLRCFGESEVSWQYPMSEEESSDVEIRNEENNSGLFVTVLEVSSASAAHTGLYTCYYNHTQ TEENELEGRHIYIYVPDPDVAFVPLGMTDYLVIVEDDDSAIIPCRTTDPETPVTLHNSEGVVPASYDSRQGFNGTFTVGPYIC EATVKGKKFQTIPFNVYALKATSELDLEMEALKTVYKSGETIVVTCAVFNNEVVDLQWTYPGEVKGKGITMLEEIKVPSIKLV YTLTVPEATVKDSGDYECAARQATREVKEMKKVTISVHEKGGGGGSGGGGSGGGGSASPAAPAPASPAAPAPSAPAASPAAPA PASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPAS PAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAA PAPSAPAGGGGSGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSR KGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSK HQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK SEQ ID No. 53: Nucleotide sequence encoding EPS1107P, excluding signal polypeptide sequence and purification-tag tctgataccggcagacccttcgtggaaatgtacagcgagatccccgagatcatccacatgaccgagggcagagagctggtcat cccctgcagagtgacctctcctaacatcaccgtgactctgaagaagttccctctggacacactgatccccgacggcaagagaa tcatctgggactcccggaagggcttcatcatctccaacgccacctacaaagagatcggcctgctgacctgcgaggccaccgtt aatggccacctgtacaagaccaactatctgacccacagacagaccaacaccatcatcgacgtggtgctgagcccctctcatgg catcgagctgtccgtgggagaaaagctggtgctgaactgcaccgccagaaccgagctgaacgtgggcatcgacttcaactggg agtacccctccagcaagcaccagcacaagaagctggtcaaccgggacctgaaaacccagtccggctccgagatgaagaaattc ctgagcaccctgaccatcgacggcgtgaccagatctgaccagggcctgtatacctgcgccgcttcctctggcctgatgaccaa gaaaaactccaccttcgtgcgggtgcacgagaaaggtggcggaggatctggcggaggcggctctggcggcggtggatctgctt ctcctgctgctccagctccagcttctccagcagctcctgcaccttctgcaccagctgcaagtcctgcagcacccgcaccagct agtcctgccgctcctgctcctagtgctcctgccgcaagtccagctgctcccgctcctgcaagcccagctgcaccagcaccaag tgctccagctgcctcaccagccgcaccagctccagcaagccctgcagctcccgctccttcagctcctgctgcttctcccgcag cacccgctccagcatcaccagccgctccagcaccatcagctccagcagcatctcctgcagctccagctcctgctagtcccgct gctcccgcacctagtgcaccagccgcttctcccgccgctcctgctcctgcatctcctgctgcacccgctccatctgctcccgc cgcatcacccgcagctcccgcaccagcctctccagctgcaccagctcctagcgcaccagcagctagcccagctgctcctgcac cagctagccccgcagctccagctccaagcgctcctgctgcatccccagctgctccagctcctgcctcaccagctgctccagca ccttctgctcccgctggcggtggcggaagcggaggtggtggtagtggcggcggaggttctcagctgtccctgccttctatcct gcctaacgagaacgagaaggtggtccagctgaactcctccttctctctgcggtgcttcggcgagtccgaagtgtcttggcagt accccatgtccgaagaggaatcctccgacgtggaaatccggaacgaggaaaacaactccggcctgttcgtgaccgtgctggaa gtgtcctctgcctctgctgctcacaccggcctgtacacatgctactacaatcacacccagaccgaagagaacgagctggaagg ccggcacatctacatctacgtgcccgatcctgacgtggcctttgtgcctctgggcatgaccgactacctggtcatcgtggaag atgacgactccgctatcatcccttgccggaccaccgatccagagacacctgtgacactgcacaactccgaaggcgtggtgcct gcctcctacgattctagacagggcttcaacggcaccttcaccgtgggaccttacatctgcgaggctacagtgaagggcaagaa gtttcagacaatccccttcaacgtgtacgccctgaaggccacctctgagctggacctggaaatggaagctctgaaaaccgtgt acaagtccggcgagacaatcgtcgtgacctgtgccgtgttcaacaacgaagtggtggacctgcagtggacctatcctggcgaa gtgaaaggcaagggcatcaccatgctggaagagatcaaggtgccctccatcaagctggtgtataccctgaccgtgcctgaggc cactgtgaaggactctggcgactacgagtgtgccgctagacaggccaccagagaagtcaaagaaatgaagaaagtgaccatct ccgtccacgagaagggc SEQ ID No. 54: Amino acid sequence of EPS1107P, excluding signal polypeptide sequence and purification-tag SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATV NGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKF LSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKGGGGSGGGGSGGGGSASPAAPAPASPAAPAPSAPAASPAAPAPA SPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPA APAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPA PSAPAGGGGSGGGGSGGGGSQLSLPSILPNENEKVVQLNSSFSLRCFGESEVSWQYPMSEEESSDVEIRNEENNSGLFVTVLE VSSASAAHTGLYTCYYNHTQTEENELEGRHIYIYVPDPDVAFVPLGMTDYLVIVEDDDSAIIPCRTTDPETPVTLHNSEGVVP ASYDSRQGFNGTFTVGPYICEATVKGKKFQTIPFNVYALKATSELDLEMEALKTVYKSGETIVVTCAVFNNEVVDLQWTYPGE VKGKGITMLEEIKVPSIKLVYTLTVPEATVKDSGDYECAARQATREVKEMKKVTISVHEKG SEQ ID No. 55: Nucleotide sequence encoding EPS1109P, excluding signal polypeptide sequence and purification-tag gcttctccagccgctccagctcctgcttctcctgctgcaccagcaccatctgctccagctgcaagtccagctgctcccgcacc agcaagtcctgcagcacccgctcctagtgctccagcagcatctcccgcagcaccagctccagcttcaccagcagctcccgctc catcagcaccagccgcatcacccgctgctccagcaccagcttctcccgccgctcctgcaccttctgcacccgcagctagccct gctgctcctgctccagcatctccagctgcacccgctccaagcgcacccgctgctagtccagcagcaccagcaccagctagtcc cgctgctccagctccttctgctccagcagcttcaccagccgctccagcaccagctagcccagccgcaccagcacctagtgctc ccgccgctagtcctgcagctccagctcctgctagcccagctgctcccgctectagcgctcctgccgcttcaccagctgcacca gctccagcaagtccagccgctcctgctccaagtgcaccagctgcctctccagctgctcctgctcctgcaagtoccgcagctcc agcacctagcgcaccagcatctgataccggcagacccttcgtggaaatgtacagcgagatccccgagatcatccacatgaccg agggcagagagctggtcatcccctgcagagtgacctctcctaacatcaccgtgactctgaagaagttccctctggacacactg atccccgacggcaagagaatcatctgggactcccggaagggcttcatcatctccaacgccacctacaaagagatcggcctgct gacctgcgaggccaccgttaatggccacctgtacaagaccaactatctgacccacagacagaccaacaccatcatcgacgtgg tgctgagcccctctcatggcatcgagctgtccgtgggagaaaagctcgtgctgaactgcaccgccagaaccgagctgaacgtg ggcatcgacttcaactgggagtaccccagctccaaacaccagcacaagaaactggtcaaccgggacctgaaaacccagtccgg ctccgagatgaagaaattectgagcaccctgaccatcgacggcgtgaccagatctgaccagggcctgtatacctgcgccgctt cttctggcctgatgaccaagaaaaactccaccttcgtgcgcgtgcacgagaagcagctgtccctgccttctatcctgcctaac gagaacgagaaggtggtccagctgaactcctccttctctctgcggtgcttcggcgagtccgaagtgtcttggcagtaccccat gtccgaagaggaatcctccgacgtggaaatccggaacgaggaaaacaactccggcctgttcgtgaccgtgctggaagtgtcct ctgcctctgctgctcacaccggcctgtacacatgctactacaatcacacccagaccgaagagaacgagctggaaggccggcac atctacatctacgtgcccgatcctgacgtggcctttgtgcctctgggcatgaccgactacctggtcatcgtggaagatgacga ctccgctatcatcccttgccggaccaccgatccagagacacctgtgacactgcacaactccgaaggcgtggtgcctgcctcct acgattctagacagggcttcaacggcaccttcaccgtgggaccttacatctgcgaggctacagtgaagggcaagaagtttcag acaatccccttcaacgtgtacgccctgaaggccacctctgagctggacctggaaatggaagctctgaaaaccgtgtacaagtc cggcgagacaatcgtcgtgacctgtgccgtgttcaacaacgaggtggtggacctgcagtggacctatcctggcgaagtgaaag gcaagggcatcaccatgctggaagagatcaaggtgccctccatcaagctggtgtataccctgaccgtgcctgaggccactgtg aaggactctggcgactacgagtgtgccgctagacaggccaccagagaagtcaaagaaatgaagaaagtgaccatctccgtcca cgagaagggc SEQ ID No. 56: Amino acid sequence of EPS1109P, excluding signal polypeptide sequence and purification-tag ASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASP AAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAP APASPAAPAPSAPAASPAAPAPASPAAPAPSAPASDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTL IPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNV GIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKQLSLPSILPN ENEKVVQLNSSFSLRCFGESEVSWQYPMSEEESSDVEIRNEENNSGLFVTVLEVSSASAAHTGLYTCYYNHTQTEENELEGRH IYIYVPDPDVAFVPLGMTDYLVIVEDDDSAIIPCRTTDPETPVTLHNSEGVVPASYDSRQGFNGTFTVGPYICEATVKGKKFQ TIPFNVYALKATSELDLEMEALKTVYKSGETIVVTCAVFNNEVVDLQWTYPGEVKGKGITMLEEIKVPSIKLVYTLTVPEATV KDSGDYECAARQATREVKEMKKVTISVHEKG SEQ ID No. 57: Nucleotide sequence encoding EPS1110P, excluding signal polypeptide sequence and purification-tag gcttctccagccgctccagctcctgcttctcctgctgcaccagcaccatctgctccagctgcaagtccagctgctcccgcacc agcaagtcctgcagcacccgctcctagtgctccagcagcatctcccgcagcaccagctccagcttcaccagcagctcccgctc catcagcaccagccgcatcacccgctgctccagcaccagcttctcccgccgctcctgcaccttctgcacccgcagctagccct gctgctcctgctccagcatctccagctgcacccgctccaagcgcacccgctgctagtccagcagcaccagcaccagctagtcc cgctgctccagctccttctgctccagcagcttcaccagccgctccagcaccagctagcccagccgcaccagcacctagtgctc ccgccgctagtcctgcagctccagctcctgctagcccagctgctcccgctcctagcgctcctgccgcttcaccagctgcacca gctccagcaagtccagccgctcctgctccaagtgcaccagctgcctctccagctgctcctgctcctgcaagtcccgcagctcc agcacctagcgcaccagctcaactgtccctgccttccatcctgcctaacgagaacgagaaggtggtccagctgaactcctcct tctctctgcggtgcttcggcgagtccgaagtgtcttggcagtaccccatgtccgaagaggaatcctccgacgtggaaatccgg aacgaggaaaacaactccggcctgttcgtgaccgtgctggaagtgtcctctgcctctgctgctcacaccggcctgtacacctg ttactacaatcacacccagaccgaagagaacgagctggaaggccggcacatctacatctacgtgcccgatcctgacgtggcct ttgtgcctctgggcatgaccgactacctggtcatcgtggaagatgacgactccgctatcatcccctgccggaccacagatcct gagacacctgtgacactgcacaactccgaaggcgtggtgcctgcctcctacgattctagacagggcttcaacggcaccttcac cgtgggaccttacatctgcgaggctaccgtgaagggcaagaagttccagacaatccccttcaacgtgtacgccctgaaggcca cctctgagctggacctggaaatggaagccctgaaaaccgtgtacaagtccggcgagacaatcgtcgtgacctgcgccgtgttc aacaacgaggtggtggacctgcagtggacctatcctggcgaagtgaaaggcaagggcatcaccatgctggaagagatcaaggt gccctccatcaagctggtgtataccctgaccgtgcctgaggccacagtgaaggactctggcgactacgagtgtgccgctagac aggccaccagagaagtcaaagagatgaagaaagtcaccatctccgtgcacgagaagggctccgataccggcagacccttcgtg gaaatgtacagcgagatccccgagatcatccacatgaccgagggcagagagctggtcatcccttgcagagtgacctctcctaa catcacagtgaccctgaagaagtttcccctggacacactgatccccgacggcaagagaatcatctgggactcccggaagggct tcatcatctccaacgccacctacaaagagatcggcctgctgacctgtgaagccaccgtgaatggccacctgtacaagaccaac tatctgacccacagacagaccaacaccatcatcgacgtggtgctgtccccaagccatggcatcgagctgtccgtgggagaaaa gctcgtgctgaactgcaccgccagaaccgagctgaacgtgggcatcgacttcaactgggagtaccccagctccaaacaccagc acaagaaactggtcaaccgggacctcaagacccagtccggctccgaaatgaagaaattcctgagcaccctgaccatcgacggc gtgaccagatctgaccagggactgtatacctgtgccgcctcctctggcctgatgaccaagaaaaactccaccttcgtgcgggt ccacgagaag SEQ ID No. 58: Amino acid sequence of EPS1110P, excluding signal polypeptide sequence and purification-tag ASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASP AAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAP APASPAAPAPSAPAASPAAPAPASPAAPAPSAPAQLSLPSILPNENEKVVQLNSSFSLRCFGESEVSWQYPMSEEESSDVEIR NEENNSGLFVTVLEVSSASAAHTGLYTCYYNHTQTEENELEGRHIYIYVPDPDVAFVPLGMTDYLVIVEDDDSAIIPCRTTDP ETPVTLHNSEGVVPASYDSRQGFNGTFTVGPYICEATVKGKKFQTIPFNVYALKATSELDLEMEALKTVYKSGETIVVTCAVF NNEVVDLQWTYPGEVKGKGITMLEEIKVPSIKLVYTLTVPEATVKDSGDYECAARQATREVKEMKKVTISVHEKGSDTGRPFV EMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTN YLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDG VTRSDQGLYTCAASSGLMTKKNSTFVRVHEK SEQ ID No. 59: Nucleotide sequence encoding EPS1111P, excluding signal polypeptide sequence and purification-tag gcttctccagccgctccagctcctgcttctcctgctgcaccagcaccatctgctccagctgcaagtccagctgctcccgcacc agcaagtcctgcagcacccgctcctagtgctccagcagcatctcccgcagcaccagctccagcttcaccagcagctcccgctc catcagcaccagccgcatcacccgctgctccagcaccagcttctcccgccgctcctgcaccttctgcacccgcagctagccct gctgctcctgctccagcatctccagctgcacccgctccaagcgcacccgctgctagtccagcagcaccagcaccagctagtcc cgctgctccagctccttctgctccagcagcttcaccagccgctccagcaccagctagcccagccgcaccagcacctagtgctc ccgccgctagtcctgcagctccagctcctgctagcccagctgctcccgctcctagcgctcctgccgcttcaccagctgcacca gctccagcaagtccagccgctcctgctccaagtgcaccagctgcctctccagctgctcctgctcctgcaagtcccgcagctcc agcacctagcgcaccagcatctgataccggcagacccttcgtggaaatgtacagcgagatccccgagatcatccacatgaccg agggcagagagctggtcatcccctgcagagtgacctctcctaacatcaccgtgactctgaagaagttccctctggacacactg atccccgacggcaagagaatcatctgggactcccggaagggcttcatcatctccaacgccacctacaaagagatcggcctgct gacctgcgaggccaccgttaatggccacctgtacaagaccaactatctgacccacagacagaccaacaccatcatcgacgtgg tgctgagcccctctcatggcatcgagctgtccgtgggagaaaagctcgtgctgaactgcaccgccagaaccgagctgaacgtg ggcatcgacttcaactgggagtaccccagctccaaacaccagcacaagaaactggtcaaccgggacctgaaaacccagtccgg ctccgagatgaagaaattcctgagcaccctgaccatcgacggcgtgaccagatctgaccagggcctgtatacctgcgccgctt cttctggcctgatgaccaagaaaaactccaccttcgtgcgcgtgcacgagaagaacgatgccgaggaactgttcatcttcctg accgagattaccgagatcacaatcccctgccgcgtgacagatcctcagctggtggttaccctgcatgagaagaaaggcgacgt ggccctgcctgtgccttacgatcatcagagaggcttctccggcatcttcgaggaccggtcttacatctgcaagaccaccatcg gcgacagagaggtggactccgacgcctactacgtgtacagactccaggtgtcctccatcaacgtgtccgtgaatgccgtgcag acagttgtgcggcagggcgagaatatcaccctgatgtgcatcgtgatcggcaacgaggtggtcaacttcgagtggacctatcc tcggaaagaatctggccggctggtggaacctgtgaccgacttcctgctggacatgccctaccacatccggtctatcctgcaca tcccttccgccgagctggaagattccggcacctacacctgtaacgtgaccgagtccgtgaacgaccaccaggacgagaaggcc atcaatatcaccgtggtggaatccggctacgtgcggctgttgggagaagtgggcacactgcagtttgctgagctg SEQ ID No. 60: Amino acid sequence of EPS1111P, excluding signal polypeptide sequence and purification-tag ASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASP AAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAP APASPAAPAPSAPAASPAAPAPASPAAPAPSAPASDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTL IPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNV GIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKNDAEELFIFL TEITEITIPCRVTDPQLVVTLHEKKGDVALPVPYDHQRGFSGIFEDRSYICKTTIGDREVDSDAYYVYRLQVSSINVSVNAVQ TVVRQGENITLMCIVIGNEVVNFEWTYPRKESGRLVEPVTDFLLDMPYHIRSILHIPSAELEDSGTYTCNVTESVNDHQDEKA INITVVESGYVRLLGEVGTLQFAEL SEQ ID No. 61: Nucleotide sequence encoding EPS1113P, excluding signal polypeptide sequence and purification-tag cagctgtccctgccttctatcctgcctaacgagaacgagaaggtggtgcagctgaactcctccttctctctgcggtgcttcgg cgagtccgaagtgtcttggcagtaccccatgtccgaagaggaatcctccgacgtggaaatccggaacgaggaaaacaactccg gcctgttcgtgaccgtgctggaagtgtcctctgcctctgctgctcacaccggcctgtacacctgttactacaatcacacccag accgaagagaacgagctggaaggccggcacatctacatctacgtgcccgatcctgacgtggcctttgtgcctctgggcatgac cgactacctggtcatcgtggaagatgacgactccgctatcatcccctgccggaccacagatcctgagacacctgtgacactgc acaactccgaaggcgtggtgcctgcctcctacgattctagacagggcttcaacggcaccttcaccgtgggaccttacatctgc gaggctaccgtgaagggcaagaagttccagacaatccccttcaacgtgtacgccctgaaggccacctctgagctggacctgga aatggaagccctgaaaaccgtgtacaagtccggcgagacaatcgtcgtgacctgcgccgtgttcaacaacgaggtggtggacc tgcagtggacctatcctggcgaagtgaaaggcaagggcatcaccatgctggaagagatcaaggtgccctccatcaagctggtg tataccctgaccgtgcctgaggccacagtgaaggactctggcgactacgagtgtgccgctagacaggccaccagagaagtcaa agagatgaagaaagtcaccatctccgtgcacgagaagggcgcctctccagctgctcctgctccagctagtcctgcagctccag ctccatctgcaccagctgcttctccagcagcacccgcaccagcttctcccgccgctcctgcacctagtgcaccagcagctagc cctgctgcaccagcaccagcaagtccagccgcaccagctcctagtgctccagctgcatcccctgctgctcccgctcctgcttc accagccgctccagcaccatcagctcccgcagcatctccagcagctccagctcctgcttctcctgctgcacccgctccatctg ctcccgctgcaagtcctgctgctcctgcaccagcatcacccgcagctcccgcaccaagcgctccagccgcttcacccgcagca ccagctccagcctcaccagcagcaccagcaccttccgctccagctgctagtccagccgctcctgctcctgcaagccccgctgc tccagctcctagcgcacccgctgctagccccgcagctcccgctccagcaagcccagcagctcctgctccttctgctccagcag catctcctgccgcaccagctccagctagcccagctgctcccgcaccatccgcaccagcagcaagtcccgcagctccagcacca gctagtcccgcagcacccgcaccttcagcaccagccgcatcaccagctgctccagctccagcatctcccgctgcaccagcacc aagtgctcccgctgcttctcctgcagctcctgctccagcctctccagctgctcccgcaccttctgctccagctgcctctccag ctgctccagcaccagcttcaccagctgctcccgctcctagtgctcctgccgctagtccagcagctcccgcaccagctagccct gccgctcctgctccaagtgctccagccgcaagtcccgctgcacccgctccagcttctccagcagctcccgctccaagcgcacc cgcagcttctcccgctgctcccgcaccagcaagtcctgctgctccagctccttcagctcctgccgcttctcctgctgctccag ctcctgcaagtccagctgctccagcaccaagtgcaccagcagcaagtccagctgctcctgctcctgcctctccagcagcacca gctcctagcgcaccagccgccagtcctgcagcaccagctccagcttctcccgctgctcctgctccttcagcaccagctgctag tcctgctgctcctgctccagcttctcctgccgctccagcaccaagcgctccagctgcatctcccgcagctcccgctccagcat ctcctgcagcacccgcaccatcagctccagctgcttccccagccgctcctgcaccagctagcccagcagctcctgcacctagc gctcccgctgcttcaccagcagctccagcaccagccagtccagctgctcctgcaccatctgcacccgctgctagtcccgctgc tccagctcctgctagccctgcagcaccagctccaagtgcacccgccgcatcacccgccgcaccagcaccagcaagccctgcag cacccgctccaagcgctccagctgctagcccagcagcaccagcaccagcatcaccagccgctccagcaccttctgcaccagca gcttcacccgctgcacccgctccagcatcacccgccgctccagctcctagcgctcctgcagcctctcctgcagctccagcacc agcaagccccgctgcaccagcaccatctgctccagcagctagccctgcagctcccgctcctgcatctcccgccgcaccagctc catctgcacccgcagcatctgataccggcagacccttcgtggaaatgtacagcgagatccccgagatcatccacatgaccgag ggcagagagctggtcatcccttgcagagtgacctctcctaacatcacagtgaccctgaagaagtttcccctggacacactgat ccccgacggcaagagaatcatctgggactcccggaagggcttcatcatctccaacgccacctacaaagagatcggcctgctga cctgtgaagccaccgtgaatggccacctgtacaagaccaactatctgacccacagacagaccaacaccatcatcgacgtggtg ctgagcccctctcatggcatcgagctgtccgtgggagagaagctcgtgctgaactgtaccgccagaaccgagctgaacgtggg catcgacttcaactgggagtaccctagctccaaacaccagcacaagaaactggtcaaccgggacctcaagacccagtccggct ccgaaatgaagaaattcctgtccacactgaccatcgacggcgtgaccagatctgaccagggactgtatacctgtgccgcctcc tctggcctgatgaccaagaaaaactccaccttcgtgcgggtccacgagaag SEQ ID No. 62: Amino acid sequence of EPS1113P, excluding signal polypeptide sequence and purification-tag QLSLPSILPNENEKVVQLNSSFSLRCFGESEVSWQYPMSEEESSDVEIRNEENNSGLFVTVLEVSSASAAHTGLYTCYYNHTQ TEENELEGRHIYIYVPDPDVAFVPLGMTDYLVIVEDDDSAIIPCRTTDPETPVTLHNSEGVVPASYDSRQGFNGTFTVGPYIC EATVKGKKFQTIPFNVYALKATSELDLEMEALKTVYKSGETIVVTCAVFNNEVVDLQWTYPGEVKGKGITMLEEIKVPSIKLV YTLTVPEATVKDSGDYECAARQATREVKEMKKVTISVHEKGASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAAS PAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAA PAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAP ASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASP AAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAP APSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPS APAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPA ASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASDTGRPFVEMYSEIPEIIHMTE GRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVV LSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAAS SGLMTKKNSTFVRVHEK SEQ ID No. 63: Nucleotide sequence encoding EPS1114P, excluding signal polypeptide sequence and purification-tag cagctgtccctgccttccatcctgcctaacgagaacgagaaggtggtgcagctgaactcctccttctctctgcggtgcttcgg cgagtccgaagtgtcttggcagtaccccatgtccgaagaggaatcctccgacgtggaaatccggaacgaggaaaacaactccg gcctgttcgtgaccgtgctggaagtgtcctctgcctctgctgctcacaccggactgtacacctgttactacaatcacacccag accgaagagaacgagctggaaggccggcacatctacatctacgtgcccgatcctgacgtggcctttgtgcctctgggcatgac cgactacctggtcatcgtggaagatgacgactccgctatcatcccctgccggaccacagatcctgagacacctgtgacactgc acaactccgaaggcgtggtgcctgcctcctacgattctagacagggcttcaacggcaccttcaccgtgggaccttacatctgc gaggctaccgtgaagggcaagaagttccagacaatccccttcaacgtgtacgccctgaaggccacctctgagctggacctgga aatggaagccctgaaaaccgtgtacaagagcggcgagacaatcgtcgtgacctgcgccgtgttcaacaacgaggtggtggacc tgcagtggacctatcctggcgaagtgaaaggcaagggcatcaccatgctggaagagatcaaggtgccctccatcaagctggtg tataccctgaccgtgcctgaggccacagtgaaggactctggcgactacgagtgtgccgctagacaggccaccagagaagtcaa agagatgaagaaagtcaccatctccgtgcacgagaaaggcggcggaggcggaagcggtggcggaggaagcggaggcggcggat ctgcttctcctgctgctcctgctccagctagtcctgctgcaccagcaccttcagctccagctgcttctccagcagcacccgca ccagcatcaccagccgctccagcaccaagtgcaccagctgctagcccagctgctcccgctcctgcatctcctgcagcaccagc tccatctgcaccagcagcaagtccagcagctccagctcctgcttcacccgctgctcccgcaccatctgctccagccgcatcac ccgctgcaccagctccagcttctcccgccgctccagctccttctgctcctgcagcatctcctgctgctccagcaccagcaagc ccagccgctcctgctccatcagcacccgctgcctctccagctgctcctgcaccagcctctccagctgcacccgctcctagtgc tccagctgcaagtcccgccgcaccagcaccagctagtcctgcagctcctgcaccaagcgctccagcagcttcccctgcagctc ctgctcctgcctctcctgccgctcctgctcctagtgcaccagccgcatctcccgcagctcccgctcctgctagtccagcagct cccgcaccttctgcaccagcagcttccccagccgcaccagctccagcaagccccgctgctccagcacctagtgctcccgctgc ctcaccagcagctcccgctccagcaagccctgctgcacccgctccaagcgcaccagcagcatcaccagctgcacccgcaccag ctagcccagcagcaccagctcctagcgctcccgcagctagccctgctgctcccgcaccagcttcacccgcagcacccgctcca tcagctcccgccgctagtcccgctgctcctgctcctgcaagccctgctgctcctgctccttctgctccagctgctagtcctgc cgctcctgctccagcttctccagcagctcctgcacctagcgcacccgccgctagtccagcagcaccagcaccagcttctccag ctgcaccagcaccatcagcacccgcagcttcaccagcagctccagcaccagcatctcccgcagctccagcaccatcagctcca gcagcaagcccagctgcaccagctccagcatcaccagctgctcccgctccaagcgctcctgctgcttctcctgccgcaccagc tccagccagtccagcagcacccgctccaagtgcacccgccgcttctccagctgctccagctcctgctagccccgcagctccag ctccaagtgctccagccgccagtcctgcagctcccgcaccagctagccccgctgctcctgcaccatccgcaccagctgctagt cccgcagcaccagctccagctagcccagccgcaccagcaccatctgctcccgctgctagccctgcagcacccgctccagccag tcctgctgctccagctccatctgctcccgccgcttctcctgcagctcctgcaccagcttctcccgctgctcctgctcctagcg ctccagcagcctctccagcagcaccagctccagcaagtcctgcagcaccagcacctagtgcaccagcagcttcacccgctgct cccgctccagcatctccagctgctccagcaccttctgctccagctgcaagccccgcagctcctgcaccagcaagtcctgccgc tccagctcctagcgctcctgctgcaagtccagctgctcccgctccagcttcaccagccgcaccagcaccttccgcaccagcag ctagtccagctgctcctgctccagctagcccagctgctccagctccttcagcaccagcagccggtggcggaggatctggcgga ggcggatctggcggcggtggttcttctgataccggcagacccttcgtggaaatgtacagcgagat ccccgagatcatccacatgaccgagggcagagagctggtcatcccttgcagagtgacctctcctaacatcacagtgaccctga agaagtttcccctggacacactgatccccgacggcaagagaatcatctgggactcccggaagggcttcatcatctccaacgcc acctacaaagagatcggactgctgacctgcgaagccactgtgaacggccacctgtacaagaccaactatctgacccacagaca gaccaacaccatcatcgacgtggtgctgagcccctctcatggcatcgagctgtccgt gggagagaaactggtgctgaactgcaccgccagaaccgagctgaacgtgggcatcgacttcaactgggagtaccccagctcca aacaccagcacaagaagctggtcaaccgggatctgaaaacccagtccggctccgaaatgaagaaattcctgagcaccctgacc atcgacggcgtgaccagatctgaccagggcctgtatacctgtgccgcctcttctggcctgatgaccaagaaaaactccacctt cgtgcgggtccacgagaag SEQ ID No. 64: Amino acid sequence of EPS1114P, excluding signal polypeptide sequence and purification-tag QLSLPSILPNENEKVVQLNSSFSLRCFGESEVSWQYPMSEEESSDVEIRNEENNSGLFVTVLEVSSASAAHTGLYTCYYNHTQ TEENELEGRHIYIYVPDPDVAFVPLGMTDYLVIVEDDDSAIIPCRTTDPETPVTLHNSEGVVPASYDSRQGFNGTFTVGPYIC EATVKGKKFQTIPFNVYALKATSELDLEMEALKTVYKSGETIVVTCAVFNNEVVDLQWTYPGEVKGKGITMLEEIKVPSIKLV YTLTVPEATVKDSGDYECAARQATREVKEMKKVTISVHEKGGGGGSGGGGSGGGGSASPAAPAPASPAAPAPSAPAASPAAPA PASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPAS PAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAA PAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAP SAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAP AASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAAS PAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAA PAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAAGGGGSGG GGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIG LLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQ SGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK SEQ ID No. 65: Nucleotide sequence encoding EPS1115P, excluding signal polypeptide sequence and purification-tag tctgataccggcagacccttcgtggaaatgtacagcgagatccccgagatcatccacatgaccgagggcagagagctggtcat cccctgcagagtgacctctcctaacatcaccgtgactctgaagaagttccctctggacacactgatccccgacggcaagagaa tcatctgggactcccggaagggcttcatcatctccaacgccacctacaaagagatcggcctgctgacctgcgaggccaccgtt aatggccacctgtacaagaccaactatctgacccacagacagaccaacaccatcatcgacgtggtgctgagcccctctcatgg catcgagctgtccgtgggagaaaagctggtgctgaactgcaccgccagaaccgagctgaacgtgggcatcgacttcaactggg agtacccctccagcaagcaccagcacaagaagctggtcaaccgggacctgaaaacccagtccggctccgagatgaagaaattc ctgagcaccctgaccatcgacggcgtgaccagatctgaccagggcctgtatacctgcgccgcttcctctggcctgatgaccaa gaaaaactccaccttcgtgcgggtgcacgagaaaggtggcggaggatctggcggaggcggctctggcggcggtggatctgctt ctcctgctgctccagctccagcttctccagcagctcctgcaccttctgcaccagctgcaagtcctgcagcacccgcaccagct agtcctgccgctcctgctcctagtgctcctgccgcaagtccagctgctcccgctcctgcaagcccagctgcaccagcaccaag tgctccagctgcctcaccagccgcaccagctccagcaagccctgcagctcccgctccttcagctcctgctgcttctcccgcag cacccgctccagcatcaccagccgctccagcaccatcagctccagcagcatctcctgcagctccagctcctgctagtcccgct gctcccgcacctagtgcaccagccgcttctcccgccgctcctgctcctgcatctcctgctgcacccgctccatctgctcccgc cgcatcacccgcagctcccgcaccagcctctccagctgcaccagctcctagcgcaccagcagctagcccagctgctcctgcac cagctagccccgcagctccagctccaagcgctcctgctgcatccccagctgctccagctcctgcctcaccagctgctccagca ccttctgctcccgccgcttctcctgccgcaccagctccagctagtccagccgcaccagcaccatctgcacccgctgctagccc tgctgcaccagctccagcatcacccgctgcaccagctccatccgcaccagctgcttcaccagcagctcccgctccagcttcac ccgctgctcccgctcctagcgctcccgcagcttcaccagctgcacccgctccagccagtccagctgctcccgcaccatccgca ccagcagcaagtcccgccgctccagctccagctagcccagctgctccagctccatctgcaccagccgcatctccagctgctcc agctccagctagtcctgctgcacccgctcctagcgctccagctgcaagtcctgccgctcctgctccagcctctcctgccgctc cagcacctagcgctcccgctgccagtccagcagctccagctcctgcatctcccgccgcaccagcaccaagcgcacccgcagca tctcccgctgctcccgctccagcaagccctgccgctcctgcaccaagtgcaccagcagcatccccagcagctcccgctccagc atctccagcagctccagctccaagtgctccagcagctagtcctgctgctccagctcctgctagccctgcagctcctgcaccat ctgctcccgcagccagtcctgcagctcctgcaccagcaagtccagctgctcctgcacctagcgctccagctgcatctcccgct gcaccagctccagcaagtcccgctgctcctgctccttctgctccagcagcttcccctgctgctcctgctcctgcttcacccgc cgctccagctccatctgctcccgctgcctctccagccgctcctgcaccagcatcaccagctgctcccgcaccaagcgcacccg ctgcaagcccagccgctcctgctcctgctagtccagccgctcctgcaccttcagcacccgcagcttccccagctgctccagct ccagcaagtccagcagctccagctccttccgctccagctgcaagccccgcagctccagctcctgcttctcctgctgctcctgc accatcagctccagctgctagtccagcagctcctgcaccagccagtcctgccgcaccagcaccttcagctccagctgcttcac ccgctgctcccgcaccagctagtccagccgctccagcaccaagtgctcccgccgctggtggtggtggatctggtggtggcgga agcggaggtggtggttctcagctgtccctgccttccatcctgcctaacgagaacgagaaggtggtccagctgaactcctcctt ctctctgcggtgcttcggcgagtccgaagtgtcttggcagtaccccatgtccgaagaggaatcctccgacgtggaaatccgga acgaggaaaacaactccggcctgttcgtgaccgtgctggaagtgtcctctgcctctgctgctcacaccggcctgtacacatgc tactacaatcacacccagaccgaagagaacgagctggaaggccggcacatctacatctacgtgcccgatcctgacgtggcctt tgtgcctctgggcatgaccgactacctggtcatcgtggaagatgacgactccgctatcatcccttgccggaccaccgatccag agacacctgtgacactgcacaactccgaaggcgtggtgcctgcctcctacgattctagacagggcttcaacggcaccttcacc gtgggaccttacatctgcgaggctacagtgaagggcaagaagtttcagacaatccccttcaacgtgtacgccctgaaggccac ctctgagctggacctggaaatggaagctctgaaaaccgtgtacaagtccggcgagacaatcgtcgtgacctgtgccgtgttca acaacgaagtggtggacctgcagtggacctatcctggcgaagtgaaaggcaagggcatcacaatgctggaagagatcaaggtg ccctccatcaagctggtgtataccctgaccgtgcctgaggccactgtgaaggactctggcgactacgagtgtgccgctagaca ggccaccagagaagtcaaagaaatgaagaaagtgaccatctccgtccacgagaagggc SEQ ID No. 66: Amino acid sequence of EPS1115P, excluding signal polypeptide sequence and purification-tag SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATV NGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKF LSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKGGGGSGGGGSGGGGSASPAAPAPASPAAPAPSAPAASPAAPAPA SPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPA APAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPA PSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSA PAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAA SPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPA APAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPA PASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAAGGGGSGGGG SGGGGSQLSLPSILPNENEKVVQLNSSFSLRCFGESEVSWQYPMSEEESSDVEIRNEENNSGLFVTVLEVSSASAAHTGLYTC YYNHTQTEENELEGRHIYIYVPDPDVAFVPLGMTDYLVIVEDDDSAIIPCRTTDPETPVTLHNSEGVVPASYDSRQGFNGTFT VGPYICEATVKGKKFQTIPFNVYALKATSELDLEMEALKTVYKSGETIVVTCAVFNNEVVDLQWTYPGEVKGKGITMLEEIKV PSIKLVYTLTVPEATVKDSGDYECAARQATREVKEMKKVTISVHEKG SEQ ID No. 67: Nucleotide sequence encoding EPS1116P, excluding signal polypeptide sequence and purification-tag cagctgagcctgccaagcatcctgcctaacgaaaatgagaaggtggtccagctgaacagctccttcagtctgagatgctttgg cgaatcagaggtgagctggcagtacccaatgtcagaggaagagtctagtgacgtggaaattaggaatgaagagaacaattcag gactgttcgtgaccgtcctggaggtgtcaagcgccagcgccgctcacaccggactgtacacatgttactataaccatactcag accgaagagaatgaactggaggggaggcacatctccatccacgtgcccgatcctgacgtggcctttgccccactgggaatgac agattacctggtcatcgtcgaggacgatgactctgccatcattccctgccgcacctcagactccgaaactcctgtgaccctgc ataacagtgagggcgtggtccccgcctcctacgattctcgacagggattcaatggcaccttcaccgtcggaccctatatctgt gaggccactgtgaagggcaagaaattccagaccattccttttaacgtgtacgcactgaaagccacatccgaactggacctgga aatggaggccctgaagactgtctataaatctggagagactatcgtggtcacctgcgccgtgttcaacaatgaagtggtcgatg cgcagtggacttaccccggcgaggtcaagggcaaagggattaccatggacgaagagatcaaggtgcctagccagaagctggtg tacaccctgacagtcccagaagccaccgtgaaggattccggggactatgagtgtgcagcccggcaggcctccagagaagtgaa ggagatgaagaaagtgacaatcagtgtccacgagaaaggagcaagccccgccgctccagcccccgcaagcccagccgcaccag caccttccgcaccagccgcctccccagcagcacccgcacccgcttcccctgccgcccccgcccctagcgcccccgccgcctcc cctgccgccccagcccccgcctctccagccgcccctgccccatctgccccagccgccagcccagccgcccccgcccctgccag ccccgccgccccagccccctccgcccctgctgcttcccctgccgcccctgccccagccagcccagctgctcctgctccaagcg cccctgctgcaagcccagctgctccagcccccgcctctcccgctgctccagctccttctgcccctgctgcttccccagctgct cccgcccctgcctctcctgctgctcctgctccctccgcccctgctgcatcccccgctgctcctgccccagcttccccagctgc acctgctccaagcgccccagctgcaagcccagctgcacctgcacctgcttcccccgctgcccctgccccaagcgcccccgccg catcccccgccgcaccagcccccgcctcacccgcagcaccagccccatcagcaccagccgcctcaccagccgcccccgcaccc gcaagtccagcagcacccgcaccatccgcccccgccgcaagcccagccgcccccgctccagcatcccctgccgcccccgcccc cagcgcccccgccgcctcccctgccgccccagcccccgcctctccagccgcccctgccccatctgccccagccgccagccccg ccgcccccgcccctgccagccccgccgccccagccccctccgcccctgctgcttcccccgccgcccctgccccagccagccca gctgctcccgctccaagcgcccccgctgcaagcccagctgctccagcccccgcctctcccgctgctccagctccttctgcccc tgctgcttcccccgctgctcccgcccccgcctctcctgctgctcccgctccctccgcccctgctgcatcccccgctgctcctg ccccagcttccccagctgcacctgctcccagcgccccagctgcaagccccgctgcacctgcacctgcttcccccgctgcccct gccccaagcgcccccgccgcctcacccgcagcccccgctccagccagccccgcagcaccagcaccctcagccccagcctcaga taccggccggccttttgtggagatgtactccgaaatccccgagatcattcacatgaccgaagggcgagagctggtcatcccat gccgggtgacaagccccaacattactgtgaccctgaagaaattccctctggatactctgatcccagacgggaagaggatcatt tgggacagccgcaaaggcttcatcatttccaatgccacatataaggaaattggcctgctgacatgcgaggccactgtgaacgg gcacctgtacaaaaccaattatctgacacatcggcagacaaacactatcattgatgtggtcctgagcccttcccatgggatcg aactgagcgtcggagagaagctggtgctgaattgtacagccagaactgaactgaacgtgggcattgacttcaattgggagtac ccctcctctaagcaccagcataagaaactggtgaatagggatctgaaaacccagtctgggagtgagatgaagaaatttctgtc taccctgacaatcgatggcgtgacacgcagtgaccaggggctgtatacttgtgcagccagttcaggcctgatgaccaagaaga acagcacatttgtccgagtccacgaaaag SEQ ID No. 68: Amino acid sequence of EPS1116P, excluding signal polypeptide sequence and purification-tag QLSLPSILPNENEKVVQLNSSFSLRCFGESEVSWQYPMSEEESSDVEIRNEENNSGLFVTVLEVSSASAAHTGLYTCYYNHTQ TEENELEGRHISIHVPDPDVAFAPLGMTDYLVIVEDDDSAIIPCRTSDSETPVTLHNSEGVVPASYDSRQGFNGTFTVGPYIC EATVKGKKFQTIPFNVYALKATSELDLEMEALKTVYKSGETIVVTCAVFNNEVVDAQWTYPGEVKGKGITMDEEIKVPSQKLV YTLTVPEATVKDSGDYECAARQASREVKEMKKVTISVHEKGASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAAS PAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAA PAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAP ASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASP AAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAP APSAPAASPAAPAPASPAAPAPSAPASDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRII WDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEY PSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK SEQ ID No. 69: Nucleotide sequence encoding PA linker gccgctcctg ctgctccagc tcctgctgcc ccagcagccc ctgccccagc tgctcctgca gcagctcccg cagccccagc acccgccgca ccagcagctc cagcccctgc agcaccagct gctgcccctg ccgcccctgc tccagccgca cccgctgcac ccgcaccagc tgccccagcc gccgcacccg cagctccagc tcccgctgct cctgctgcac cagcccctgc cgctccagca gccgcaccag cagcaccagc cccagctgct cccgctgctc cagcacccgc agcccccgca gcagcaccag ccgctcctgc tcctgccgcc ccagcagctc ctgctccagc agcccctgct gctgctccag cagcaccagc accagctgct ccagctgccc cagctcctgc agcacccgcc gctgctcccg cagctcctgc ccctgctgca cccgcagcac ccgctccagc agcacctgca gctgcaccag ctgctcccgc acctgccgct cccgcagctc ccgctcctgc agctccagcc gcagctcctg ctgctcctgc accagcagct cccgccgcac cagctccagc tgcccctgct SEQ ID No. 70: Amino acid sequence of PA linker AAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPA AAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPA AAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPA AAPAAPAPAAPAAPAPAAPA

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All references cited herein are fully incorporated by reference. Having now fully described the invention, it will be understood by a person skilled in the art that the invention may be practiced within a wide and equivalent range of conditions, parameters and the like, without affecting the spirit or scope of the invention or any embodiment thereof. 

The invention claimed is:
 1. A protein consisting essentially of: (i) an extracellular domain of the human Platelet-Derived Growth Factor receptor (PDGFR); (ii) an extracellular domain of the human Vascular Endothelial Growth Factor receptor (VEGFR); and (iii) a linker attaching said domain of PDGFR and said domain of VEGFR, wherein the linker (a) consists of proline, alanine, and serine, or (b) consists of proline and alanine.
 2. The protein of claim 1, wherein when the linker consists of proline, alanine, and serine, said proline residues constitute more than 4% and less than 40% of said linker.
 3. The protein of claim 2, wherein said linker comprises an amino acid sequence as follows: (ASPAAPAPASPAAPAPSAPA (SEQ ID NO: 71))n, wherein n is an integer of 10-100, 10-60, 10-40, or 10-30, or wherein n is 10, 20, or
 30. 4. The protein of claim 2, wherein said linker has an amino acid sequence as shown in SEQ ID No. 2 or wherein said linker is a polypeptide encoded by a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID No.
 1. 5. The protein of claim 1, wherein when the linker consists of proline and alanine, said proline residues constitute more than about 10% and less than about 75% of said linker.
 6. The protein of claim 5, wherein said linker has an amino acid sequence as follows: (AAPAAPAPAAPAAPAAPA (SEQ ID NO: 72))n, wherein n is an integer of 10-100.
 7. The protein of claim 5, wherein said linker has an amino acid sequence as shown in SEQ ID No. 70 or wherein said linker is a polypeptide encoded by a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID No.
 69. 8. The protein of claim 2, wherein said linker has an amino acid sequence consisting of about 50 to about 3000 amino acid residues.
 9. The protein of claim 1, wherein said domain of PDGFR comprises one or more of Ig domains 1 to 5 of PDGFR, one or more of Ig domains 1 to 3 of PDGFR, or wherein said domain of PDGFR comprises Ig domains 1 to 3 of PDGFR.
 10. The protein of claim 1, wherein said domain of PDGFR is capable of binding to human Platelet-Derived Growth Factor (PDGF), optionally wherein said PDGF is a PDGF dimer, wherein said PDGF dimer is a PDGF homodimer or a PDGF heterodimer.
 11. The protein of claim 1, wherein said PDGFR is human PDGFR-α.
 12. The protein of claim 1, wherein said domain of PDGFR comprises: (a) a protein having an amino acid sequence as shown in SEQ ID No. 4 or SEQ ID No. 20; (b) a polypeptide encoded by a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID No. 3 or SEQ ID No. 19; (c) a polypeptide having an amino acid sequence encoded by a nucleic acid hybridizing under stringent conditions to the complementary strand of nucleic acid molecules as defined in (b); and (d) a polypeptide having an amino acid sequence encoded by a nucleic acid being degenerate as a result of the genetic code to the nucleotide sequence of a nucleic acid as defined in (b) or (c).
 13. The protein of claim 11, wherein said domain of PDGFR is capable of binding to Platelet-Derived Growth Factor (PDGF), wherein said PDGF is a PDGF homodimer, and wherein said PDGF homodimer is a PDGF-A homodimer, a PDGF-B homodimer, or a PDGF-C homodimer; or wherein said domain of PDGFR is capable of binding to Platelet-Derived Growth Factor (PDGF), wherein said PDGF preferably is a PDGF heterodimer, and wherein said PDGF heterodimer preferably is a heterodimer of PDGF-AB.
 14. The protein of claim 1, wherein said PDGFR is human PDGFR-β.
 15. The protein of claim 1, wherein said domain of PDGFR comprises: (a) a protein having an amino acid sequence as shown in SEQ ID No. 6; (b) a polypeptide encoded by a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID No. 5; (c) a polypeptide having an amino acid sequence encoded by a nucleic acid hybridizing under stringent conditions to the complementary strand of nucleic acid molecules as defined in (b); and (d) a polypeptide having an amino acid sequence encoded by a nucleic acid being degenerate as a result of the genetic code to the nucleotide sequence of a nucleic acid as defined in (b) or (c).
 16. The protein of claim 14, wherein said domain of PDGFR is capable of binding to Platelet-Derived Growth Factor (PDGF), wherein said PDGF preferably is a PDGF homodimer, and wherein said PDGF homodimer preferably is a PDGF-B homodimer.
 17. The protein of claim 1, wherein said domain of VEGFR comprises one or more of Ig domains 1 to 7 of VEGFR, wherein said domain of VEGFR comprises Ig domain 2 and/or Ig domain 3 of VEGFR, or wherein said domain of VEGFR comprises Ig domain 2 and Ig domain 3 of VEGFR.
 18. The protein of claim 1, wherein said VEGFR is human VEGFR-1 or human VEGFR-2, wherein said domain of VEGFR comprises Ig domain 2 of VEGFR-1 and Ig domain 3 of VEGFR-2.
 19. The protein of claim 1, wherein said domain of VEGFR comprises: (a) a protein having an amino acid sequence as shown in SEQ ID No. 8; (b) a polypeptide encoded by a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID No. 7; (c) a polypeptide having an amino acid sequence encoded by a nucleic acid hybridizing under stringent conditions to the complementary strand of nucleic acid molecules as defined in (b); and (d) a polypeptide having an amino acid sequence encoded by a nucleic acid being degenerate as a result of the genetic code to the nucleotide sequence of a nucleic acid as defined in (b) or (c).
 20. The protein of claim 1, wherein said domain of VEGFR is capable of binding to human Vascular Endothelial Growth Factor (VEGF), optionally wherein said Vascular Endothelial Growth Factor (VEGF) is a VEGF dimer, such as a VEGF homodimer or a VEGF-A homodimer.
 21. The protein of claim 1, wherein said protein is a fusion protein.
 22. The protein of claim 1, wherein said protein comprises: (a) a protein having an amino acid sequence as shown in SEQ ID No. 16, SEQ ID NO: 46, SEQ ID No. 48, SEQ ID No. 50, EQ ID No. 52, SEQ ID No.54, SEQ ID No. 56, SEQ ID No. 58, SEQ ID No. 60, SEQ ID No. 62, SEQ ID No. 64, SEQ ID No. 66 or SEQ ID No. 68; (b) a polypeptide encoded by a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID No. 15, SEQ ID No. 45, SEQ ID No. 47, SEQ ID No. 49, SEQ ID No. 51, SEQ ID No. 53, SEQ ID No. 55, SEQ ID No. 57, SEQ ID No. 59, SEQ ID No. 61, SEQ ID No. 63, SEQ ID No. 65 or SEQ ID No. 67; (c) a polypeptide having an amino acid sequence encoded by a nucleic acid hybridizing under stringent conditions to the complementary strand of nucleic acid molecules as defined in (b); and (d) a polypeptide having an amino acid sequence encoded by a nucleic acid being degenerate as a result of the genetic code to the nucleotide sequence of a nucleic acid as defined in (b) or (c).
 23. The protein of claim 1, wherein said protein comprises an N-terminal signal sequence, wherein said N-terminal signal sequence is the N-terminal signal sequence of PDGFR, such as the N-terminal signal sequence of human PDGFRα.
 24. The protein of claim 23, wherein said N-terminal signal sequence has an amino acid sequence as shown in SEQ ID No. 10 or wherein said N-terminal signal sequence is a polypeptide encoded by a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID No.
 9. 25. The protein of any claim 1, wherein the protein is arranged from N-terminus to C-terminus in the order: (optional signal sequence)-one or more domains of PDGFR-(PAS/PA)-one or more domains of VEGFR-(optional purification tag) or (optional signal sequence)-one or more domains of VEGFR-(PAS/PA)-one or more domains of PDGFR-(optional purification tag) or (optional signal sequence)-(PAS/PA)-one or more domains of VEGFR-one or more domains of PDGFR-(optional purification tag) or (optional signal sequence)-(PAS/PA)-one or more domains of PDGFR-one or more domains of VEGFR-(optional purification tag) or (optional signal sequence)-(PAS/PA)-one or more domains of PDGFR-(PAS/PA)-one or more domains of VEGFR-(PAS/PA)-(optional purification tag); or wherein the protein is arranged from N-terminus to C-terminus in the order: (optional signal sequence)-one or more domains of PDGFR-(GGGGS (SEQ ID NO: 73))n-(PAS/PA)-(GGGGS (SEQ ID NO: 73))n-one or more domains of VEGFR-(optional purification tag) or (optional signal sequence)-one or more domains of VEGFR-(GGGGS (SEQ ID NO: 73))n-(PAS/PA)-(GGGGS (SEQ ID NO: 73))n-one or more domains of PDGFR-(optional purification tag); wherein, n=0-5.
 26. The protein of claim 1, wherein said protein comprises: (a) a protein having an amino acid sequence as shown in SEQ ID No. 14, SEQ ID No. 22, SEQ ID No. 24, SEQ ID No. 26, SEQ ID No.28, SEQ ID No.30, SEQ ID No. 32, SEQ ID No. 34, SEQ ID No. 36, SEQ ID No. 38, SEQ ID No. 40, SEQ ID No. 42 or SEQ ID No. 44; (b) a polypeptide encoded by a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID No. 13, SEQ ID No.21, SEQ ID No.23, SEQ ID No.25, SEQ ID No. 27, SEQ ID No. 29, EQ ID No. 31, SEQ ID No. 33, SEQ ID No. 35, SEQ ID No. 37, SEQ ID No. 39, SEQ ID No. 41 or SEQ ID No. 43; (c) a polypeptide having an amino acid sequence encoded by a nucleic acid hybridizing under stringent conditions to the complementary strand of nucleic acid molecules as defined in (b); and (d) a polypeptide having an amino acid sequence encoded by a nucleic acid being degenerate as a result of the genetic code to the nucleotide sequence of a nucleic acid as defined in (b) or (c).
 27. A nucleic acid molecule encoding the protein of claim
 1. 28. A vector comprising the nucleic acid of claim
 27. 29. A host cell comprising the nucleic acid of claim
 27. 30. A pharmaceutical composition comprising the protein of claim 1, optionally further comprising (a) pharmaceutical acceptable carrier(s).
 31. A method for treating ophthalmic diseases, cancer, renal fibrosis, cirrhosis, arthosclerosis, portal hypertension or systemic sclerosis comprising administering the protein of claim 1 wherein said cancer is a solid cancer or non-solid cancer, optionally wherein said solid cancer is colon cancer, hepatocellular carcinoma, non-small cell lung cancer, soft tissue sarcoma, prostate cancer, breast cancer, ovarian cancer, glioma, dermatofibrosarcoma protuberans, oral squamous cell carcinoma or pancreatic cancer, optionally wherein said non-solid cancer is leukemia or non-Hodgkin's lymphoma, optionally, wherein said ophthalmic diseases is age-related macular degeneration (AMD), Diabetic retinopathy (DR), Diabetic macular edema (DME), Choroidal neovascularization (CNV), Retinal vein occlusion (RVO), Central retinal vein occlusion (CRVO), Branch retinal vein occlusion (BRVO), pathologic myopia (PM). 